food additive carrageenan: part i: a critical review of...
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http://informahealthcare.com/txcISSN: 1040-8444 (print), 1547-6898 (electronic)
Crit Rev Toxicol, Early Online: 1–33! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10408444.2013.861797
REVIEW ARTICLE
Food additive carrageenan: Part I: A critical review of carrageenanin vitro studies, potential pitfalls, and implications for human healthand safety
James M. McKim
CeeTox, Inc., Kalamazoo, MI, USA
Abstract
Carrageenan (CGN) has been used as a safe food additive for several decades. Confusion overnomenclature, basic CGN chemistry, type of CGN tested, interspecies biology, and misinter-pretation of both in vivo and in vitro data has resulted in the dissemination of incorrectinformation regarding the human safety of CGN. The issue is exacerbated when mechanisticdata obtained from in vitro experiments are directly translated to human hazard and used forrisk assessment. This can lead to information that is taken out of experimental context andreported as a definitive effect in humans. In recent years, the use of cell-based models hasincreased and their ability to provide key information regarding chemical or drug safety is wellestablished. In many instances, these new alternative approaches have started to replace theneed to use animals altogether. In vitro systems can be extremely useful for understandingsubcellular targets and mechanisms of adverse effects. However, care must be exercised whenextrapolating the in vitro findings to in vivo effects. Often, issues such as chemical identity andpurity, relevant dose, pharmacokinetic properties, solubility, protein binding, adsorption toplastics, and the use of cell models that are biologically and mechanistically relevant areoverlooked or ignored. When this occurs, in vitro findings can provide misleading informationthat is not causally linked to in vivo events in animals or in humans. To date, there has not beena comprehensive review of the CGN in vitro literature, which has reported a wide range ofbiochemical effects related to this compound. An extensive effort has been made to evaluate asmuch of this literature as possible. This review focuses on the in vitro observation, the uniquechemistry of CGN, and potential pitfalls of in vitro models used for hazard identification. Thediscussion of the in vitro studies discussed this review are supported by numerous in vivostudies. This provides a unique opportunity to have both the in vitro and in vivo studiesreviewed together.
Keywords
Carrageenan, gastrointestinal, glucosetolerance, human intestinal epithelial cells,diabetes, in vitro, NCM460, poligeenan,toll-like receptor
History
Received 6 May 2013Revised 30 October 2013Accepted 30 October 2013Published online 23 January 2014
Table of Contents
Abstract ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 1Introduction ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 1
Sources and commercial applications of carrageenan ... ... ... ... 1Importance of test article identification and purity ... ... ... ... ... ... 2CGN versus poligeenan ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 2Species differences of the GI tract anatomical, physiological, and
functional ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 3Enzymatic degradation of CGN ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 6In vitro studies and their relevance to human hazard and risk
assessment ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 9CGN and effects on the GI tract of animals ... ... ... ... ... ... ... ... ... 10Effects of CGN on glucose metabolism ... ... ... ... ... ... ... ... ... ... ... 10
In vitro studies and CGN effects on cell signaling pathways ... ... 13Effects of CGN on Wnt and bone morphogenetic protein (BMP)
signaling pathways in NCM460 cells ... ... ... ... ... ... ... ... ... ... ... 13Effects of CGN identified with various cell lines ... ... ... ... ... ... ... 18Cell-cycle arrest, cell proliferation, and cytotoxicity ... ... ... ... ... ... 18
Innate mucosal immunity and proinflammatory signalingin vitro ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 19
Effects of CGN on sulfatase activity ... ... ... ... ... ... ... ... ... ... ... ... ... 24Relationship among CGN, myoepithelial cells, and mammary
carcinoma ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 27Summary and conclusions ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 29Acknowledgements ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 30Declaration of interest ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 30References ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 30
Introduction
Sources and commercial applications of carrageenan
Carrageenan (CGN) belongs to a group of viscosifying
polysaccharides that are extracted from certain species of red
seaweeds in the family Rhodophyceae. CGN is composed of a
linear backbone of galactose sugars that have varying amounts
of sulfate attached. CGN structure can vary by conformation
and degree of sulfation. The three major forms of CGN are �-,
k-, and i-CGN. CGN is currently used primarily as a gelling,
thickening, and stabilizing agent. Figure 1 shows the structure
Address for correspondence: James M. McKim, IONTOX, LLC, Ownerand Principal, 4025 Bronson Boulevard, Kalamazoo, MI 49008, USA.E-mail: [email protected]
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of k-CGN and chondroitin-4-sulfate a molecule with a similar
structure. The food industry has been using CGN in dairy
products, such as ice cream, milk, cottage cheese, whipped
cream, yogurt, and jellies for over 50 years. This molecule has
been used for several decades as a food additive and is
considered to be safe for human consumption. This classifi-
cation was initially based on its high molecular weight, low
absorption from the gastrointestinal (GI) tract, and negative
findings in early animal safety studies (Cohen & Ito, 2002).
In this Cohen’s & Ito’s (2002) review, a comprehensive
evaluation of currently available rodent bioassay data was
discussed. However, discussions of non-animal approaches
for testing CGN safety were not discussed. Therefore, the
purpose of this review is to focus on a large number of recent
publications in which CGN was tested using non-animal or
in vitro approaches. The discussions that follow are important
because they demonstrate the importance of in vitro data as
well as the limitations associated with these studies. It is
especially important to understand the physical and chemical
properties of the test material used in vitro (e.g. CGN) as this
can significantly impact the interpretation of the data
obtained. This represents the first comprehensive review on
the many in vitro studies conducted with CGN.
Importance of test article identification and purity
In all toxicology safety studies, a critical component is the
identification and purity of the test material. This becomes
even more important when plant or seaweed extracts are the
source of the test material. Without knowing what material is
being administered, there is considerable risk for misinter-
pretation of the data obtained. The analytical protocols for
determining the average molecular weight (Mw) of CGN
requires a means to separate the molecules, such as size
exclusion chromatography (SEC) in combination with a
method of detecting concentration, such as refractive index
(RI), and a method for measurement of molecular weight,
such as light scattering (LS). It is also important to understand
that CGN can be standardized by the addition of sugars. This
point is emphasized by a recent analysis of �-CGN, which
was purchased from a laboratory supply house (Table 1).
In this case, the sample was labeled as �-CGN; however,
analysis showed that it contains only 26% �-CGN.
The remaining material was composed of 38% k-CGN and
36% sugar.
CGN versus poligeenan
There has been much confusion in the literature between the
food additive CGN and a different molecule poligeenan,
which was formerly known as ‘‘degraded CGN.’’ For the
purposes of this review, degraded CGN will be referred to as
poligeenan. The United States Adopted Names Council
(USAN, 1988) assigned the name ‘‘poligeenan’’ to the
substance previously referred to as ‘‘degraded CGN’’ to
improve issues of clarity in the scientific community.
Poligeenan is produced by subjecting CGN to acid hydrolysis
at low pH (0.9–1.3) at high temperatures (480 �C) for
extended periods of time. USAN (1988) defines poligeenan
as having a molecular weight 10 000–20 000 Da. It is used in
medical imaging, but is not a food additive and has no utility
in food. The purity/molecular weight profiles and toxico-
logical properties of CGN and poligeenan are very different.
Based on the pH of the stomach, intestinal enzymes, the form
of CGN, and the vehicle used, it is possible that some
breakdown would occur in the GI tract. However, CGN has
not been shown to be broken down in the GI tract to
poligeenan by acid hydrolysis or by microflora, and it is
poligeenan that has been shown to be responsible for
inflammatory responses in the intestine.
The molecular weight distribution of food-grade CGN
is important to discuss because issues related to toxicity
have been observed when using a low molecular weight
(520 000 Da) fraction produced by acid hydrolysis of CGN
known as poligeenan. CGN is assembled enzymatically in red
seaweed one sugar (galactose) unit at a time and, when CGN
is extracted, the sample consists of a distribution of partial
and complete CGN fragments. Commercial CGN has a Mw
range between 200 000 and 800 000 Da, but there are also
natural CGN fragments in the low molecular weight range
of 20 000–50 000 Da and also minor levels in the extremely
high molecular weight range up to 1 500 000 Da. Thus, the
molecular weight distribution of extracted CGN consists
primarily of a 200 000–800 000 Da fraction and a minor
fraction containing low molecular weight forms. It is
important to note that the composition of the low molecular
Figure 1. Molecular structure ofk-carrageenan and chondroitin-4-sulfate.
AcNH
Chondroitin-4-sulfate
HO
K-Carrageenan
HO
OH
OH
OH
OH
O
−O3SO
−O3SO−O3SO
−O3SO
OO
O
O OO O
OO
O
OH
OOO
OOO
OH
HOHO
AcNHHO
HO OOO
−O2C−O2C
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weight tail is much different when CGN is subjected to acid
hydrolysis (Panaras & Martin, 1985). Under these chemical
conditions of pH 0.9–1.3 at elevated temperatures, the
majority of the molecules are reduced to fragments that
have molecular weights below 20 000 Da. This low molecular
weight product group has been named poligeenan, and it is
this acid degradation product and NOT the naturally
occurring low molecular weight forms of CGN that are
associated with toxicity (Panaras & Martin, 1985).
Unfortunately, these names have been used interchangeably
in the literature, resulting not only in confusion, but unfounded
research conclusions. In addition, many researchers have
justified the study of CGN toxicity on the incorrect assumption
that poligeenan can be formed from the biological breakdown
of CGN in the GI tract of animals and humans. To our
knowledge, the formation of poligeenan in animals or in
humans has not been demonstrated. Therefore, it is premature
to assign liability to native CGN being converted to poligeenan
without data demonstrating that this occurs in vivo.
Species differences of the GI tract anatomical,physiological, and functional
The effects of CGN and poligeenan have been studied in
animals by administering the compounds to test animals in
diet or drinking water. Before data collected in animals can be
used to predict potential adverse events in humans, it is
important to understand the anatomical, physiological, and
functional similarities and differences between the animal
model and humans. Laboratory animals such as rats, mice,
dogs, and monkeys are commonly used as surrogates for
humans in studies intended to identify human safety issues.
Table 1. Comparison of a certificate of analysis to a detailed chemical analysis.
Sigma certificate of analysisProduct name Lambda-carrageenan (Sigma-Aldrich, BioChemika, St. Gallen, Switzerland)Product number 22049Lot number 1408463V (16 September 2008)Product brand SigmaMolecular formula Sulfated polygalactanCAS number 9064-57-7County of origin Philippines
Test Specification Results
Appearance White to light yellow powder Faintly beige powderSolubility Colorless to faintly yellow Almost colorless (10 mg/mL water)
Clear to slightly turbid (5100 NTU) Slightly turbid (30–100 NTU)Infrared spectrum Conforms to structure ConformsBio-tests Non-gelling at 1% in 0.2 M KCl Corresponds
FMC BioPolymer: chemical identity and purity analysis
Test Method Results
CompositionPure carrageenan EDTA/IPA recovery 51.3% (confirms standardizing agent levels)Potassium chloride Chloride¼ 0.4% 0.8%Calcium sulfate Free sulfate¼ 0.2% 0.3%Moisture 4 h at 105 �C 10.5%Protein Nitrogen¼ 0.1% 0.6%Diluent By difference from 100% 36.5% (sucrose or dextrose)
Total sulfate Acid digestionþBaCl2 19.0%Free sulfate BaCl2 0.2%Ester sulfate By difference 18.8% (confirms standardizing agent levels)Sodium ICP 4.5%Potassium ICP 1.0%Calcium ICP 0.4%Magnesium ICP 0.2%Arsenic MS 0.7 ppmLead MS 0.9 ppmMercury Cold vapor 0.0 ppmCadmium MS 0.3 ppmAcid insoluble matter Acid digestionþ filtration 0.2%Ash Combustion at 550 �C for 1 h 15.3% (confirms ashless standardizing agent)Acid insoluble ash Ashþ acidþ filtration 0.2%Viscosity/pH 1.5%, 75 �C, Brookfield Viscometer
(Middleboro, MA)336 m Pa s�1/9.7
Gel/non-gel KCl fractionation 59%/41% (confirms not pure lambda)Molecular weight SEC/LALS/RALS Mw¼ 1054 kDa, Mn¼ 419 kDaFTIR Film Confirms mix of unmodified kappa with
high 2-sulfate and lambda carrageenans
This product from Sigma (St. Louis, MO) is not pure lambda carrageenan as it has been blended with 36% sugar or dextrose. Also, this ‘‘lambdacarrageenan’’ comprises 59% unmodified kappa-2 with only 41% actual lambda carrageenan. This means that the actual lambda-carrageenan contentof this product is only 26%.
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In the case of orally administered test compounds, it is
essential to understand that there are fundamental differences
between humans and rodents that can profoundly affect the
overall fate of ingested material. This includes, but is not
limited to, transit time, protein binding, absorption, digestion,
and both beneficial and adverse effects. Therefore, before
reviewing specific pieces of experimental work, it is import-
ant to review the digestive systems of rodents (rats and mice)
and compare these to the human digestive tract. In order for
data obtained in rodents to be extrapolated to events predicted
to occur in humans, these differences must be incorporated
into the analysis process used to predict human risk.
The alimentary canal, which begins at the mouth and ends
at the anus, is essentially a tube lined with epithelium.
Ingested material in the alimentary canal is considered to be
outside the body. A compound cannot be considered in the
body until it actually is absorbed across the epithelium, enters
portal circulation, which supplies the liver, moves through the
liver and into systemic circulation. Therefore, in order for a
compound to affect organs inside the body, such as the liver or
heart, the agent must cross the intestinal mucous layer,
epithelium, lamina propria, and the walls of the blood and
lymph capillaries (DeSesso & Jacobson, 2001). The mechan-
isms of uptake across these natural barriers include passive
diffusion, facilitated diffusion, active transport, and pinocyt-
osis. These absorptive processes are optimized for low
molecular weight molecules, such as glucose monomers, or
low molecular weight drugs. Large molecules, such as
polysaccharides, can be divided into two types: structural
and energy storing. These macromolecules when composed
of repeating monomers of the same type linked by a
glycosidic bond are called homoglycans and if the individual
units are different they are called heteroglycans. Examples of
structural polysaccharides include pectin, cellulose, and
chitin. Humans and rodents (mice and rats) cannot digest or
break down structural polysaccharides. Energy-storing poly-
saccharides include starch and glycogen. These compounds
are made of many glucose molecules in chains of varying
lengths and can be enzymatically broken down in mammals to
allow glucose units to be released and used for energy. These
large molecules are not absorbed across intestinal barriers
without first being broken down enzymatically. Amylase is
the primary enzyme in humans and rodents that breaks down
energy-storing polysaccharides to disaccharides (maltose) and
trisaccharides (maltotriose), which must be converted to
monosaccharides in order to be absorbed in the intestine.
The three monomer sugars absorbed by the intestine are
glucose, fructose, and galactose. The recognition of polysac-
charides by digestion enzymes is based primarily on the type
of glycoside bond. CGN is formed in the cell wall of red algae
and is considered to be a structural polysaccharide, which
consists of very unique glycosidic bonds that are not
recognized by amylase or by the enzymes in gut microflora.
This reduces enzymatic degradation of CGN in the intestine
of humans and rodents.
The stomach of rodents is characterized by having two
distinct functional regions, the forestomach and glandular
stomach (Figure 2A) (DeSesso & Jacobson, 2001). In the rat,
food enters into the forestomach, which has no glandular
activity, but possesses a thick epithelium, which is a
significant barrier to absorption. The forestomach contains
bacteria that aid in the breakdown of food. Food then enters
the glandular portion of the stomach where acid is secreted
and where additional breakdown and absorption can occur.
In rats and mice, the pH of this region of the stomach ranges
from 3 to 5. This higher pH allows bacteria in the stomach to
live and participate in the digestion and absorption of food.
In contrast, the human stomach is entirely glandular with a pH
that is more acidic ranging from 1 to 2 (Figure 2B) in the
fasted state and from 4 to 5 in the presence of food (Kararli,
1995). This lower pH kills bacteria and, therefore, prevents
bacterial participation in the breakdown and absorption of
ingested material in the stomach (DeSesso & Jacobson, 2001).
The human stomach’s relative surface area is 4000 times less
than the surface area of the human small intestine, and
therefore the human stomach is a site of low absorptive
capacity. This is especially true for ionized molecules. One
exception occurs when weak acids are protonated (neutral) at
low gastric pH, which enables a higher rate of absorption.
In comparison, the surface area of the rat’s stomach is only
53 times less than the small intestine and, therefore, can
contribute to a greater proportion of absorption. In both
rodents and humans, the remaining portions of the intestine
are maintained at a pH between 7 and 8.
The time that food stays in any given portion of the GI tract
is known as the transit time and longer transit times can
increase absorption efficiency. The amount of time that it
takes for a meal to pass through the stomach is about 3–4 h in
rats and humans. This fact will be important later when the
acid hydrolysis of CGN is discussed.
In humans, most absorption occurs in the duodenum and
proximal half of the jejunum (Figure 3A). In rats, absorption
begins in the stomach and then continues in the small
intestine which is made up almost entirely of the jejunum
(Figure 3B).
The bacterial content of the human intestine does not
become significant until the ileum or the distal part of the
small intestine, while in rats and mice bacteria are present in
all portions of the GI tract. This is augmented even more by the
fact that, relatively speaking, the cecum in rats is considerably
larger than the cecum in humans and this increases the surface
area for bacteria and hence the possibility of increased
absorption in rodents due to bacterial degradation. This is
especially true when one takes into account that the large
intestinal surface area of rats is 4.5 times larger than the large
intestine of humans when expressed as a relative surface area
(DeSesso & Jacobson, 2001).
The side of the intestine that is in direct contact with the
lumen is the mucosa layer. This layer is composed of
epithelial cells (EC) and these cells are in constant flux.
Turnover of these cells occurs every 2–3 d (Creamer, 1967).
This means that cells in the small intestine mucosal layer are
constantly in a proliferative state. Figure 4 is a diagrammatic
representation of the GI tract showing the villi and crypt cells.
As stem cells in the crypt mature and migrate upto the villi,
cells slough off into the lumen.
The intestinal mucosa is complex and consists of many
types of cells with different functions. The goblet cells
produce a protective mucous film; M cells transport antigens
from the lumen to Peyer’s patches, which then allow the
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innate immune system to take action. Intestinal macrophages
can take up bacteria without themselves being activated.
T-cells deeper inside can recognize antigens presented by the
major histocompatibility complex (MHC) molecules
(Brenchley & Douek, 2008). Damage can occur when chem-
icals physically injure cells or induce the immune response of
the GI tract. This can lead to infiltration of activated T-cells and
subsequent death of villi cells, stimulation of proinflammatory
mediators, such as tumor necrosis factor alpha (TNF-a), IL-12,
and IL-8, which can lead to intestinal pathology and disease,
such as Crohn’s disease (Figure 5). In order for a chemical to
elicit these responses in the GI tract, it must cross the mucosal
layer and the EC lining the lumen.
The above discussion has pointed out some important
anatomical and physiological differences between the rodent
and the human GI tract. In addition to the anatomical and
physiological differences between the rodent and human GI
tract, there are also some important behavioral differences
between rodents and humans. The most relevant of which is
that both mice and rats practice significant coprophagia, or
eating of their feces. Mice are often housed in plastic bottom
cages in groups of 3 or more, which provides ample
opportunity to consume not only their own feces, but the
feces of their litter mates. This is extremely significant
because it means that ingested material that is poorly
absorbed can be re-ingested along with the bacteria excreted,
which can increase degradation and effectively change the
actual amount of chemical that the test animal receives
(Harmuth-Hoene & Schwerdtfeger, 1979).
Although it is has been reported that CGN administered in
drinking water of rats can be absorbed from the intestinal tract
in animal studies, the amount was considered to be extremely
low, and there is no evidence of absorption in humans
(Nicklin & Miller, 1989; Weiner, 1988, see Weiner, accom-
panying publication, 2013). Moreover, in at least two studies
by Nicklin & Miller (1984, 1983) in which CGN was provided
in drinking water, there were no intestinal lesions observed in
studies lasting as long as 90 d. In another 90-d rat study,
conducted in compliance with good laboratory practices
(GLP), in which k-CGN was added to diet, there were no
measurable effects observed, including no histopathological
findings (Weiner et al., 2007). Some rat studies in which
commercially available CGN was provided to rodents in
drinking water did result in an increase in thymidine kinase
(TK) activity (an indicator of cell proliferation) in the GI tract
(Calvert and Reicks, 1988; Calvert & Satchithanandam, 1992;
Wilcox et al., 1992). In one of these studies, CGN in the diet
increased colonic cell proliferation by five-fold, but there
were no histological changes observed (Calvert & Reicks,
1988). In a later study by Calvert & Satchithanandam (1992),
which was designed in a manner similar to their previous
study done in 1988, an increase in cell proliferation was only
Figure 2. Diagrammatic representation of ratand human stomachs. Key anatomical andfunctional differences exist in rat (A) com-pared to human (B) stomachs. In rat, thestomach is divided into the forestomach,where food enters and the glandular stomachwhere acid is produced. The forestomach hasa higher pH, which allows bacteria to live andto participate in digestion. In humans, thestomach has only a glandular function. ThepH in human stomachs is much lower,typically between 1 and 3. Bacteria cannotlive in this low pH environment. Weak acidscan have high absorption rates at low pH.Source: DeSesso et al. (2001) Food & ChemTox, 39:212
Esophagus(A)
(B)
Pylorus
Limiting ridge
Forestomach
Glandular stomach
Rugae
Esophagus
Pylorus
Rugae
Duodenum
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observed at the highest dosage administered, which was
estimated to be 100 times more than the maximum human
intake, and there were no histological abnormalities seen at
any of the doses. One explanation for different effects with
different CGN formulations (drinking water versus diet) is
that CGN used in food is less available for interaction with the
intestinal epithelium due to tight association with proteins
in the food matrix and a decreased transit time; both of
which would reduce exposure to intestinal cells. For details
on these in vivo studies, see the accompanying review by
Weiner (2013).
Enzymatic degradation of CGN
It is generally recognized by the scientific community that
intact CGN is not degraded by gut microflora to poligeenan
because intestinal enzymes do not recognize the unique
alternating a-(1-3) and b-(1-4) glycosidic bonds of CGN, and
carragenases and galactosidases are not present in the human
GI tract or microflora (JECFA, 1999 [see Weiner, Part II,
2014]; Weiner et al., 1988). In animals and humans, amylase
in the GI tract is important for the digestion of energy storing
polysaccharides, such as starch. This enzyme recognizes the
a-1–4 glycosidic bond of starch, but not the a-(1-3) or b-(1-4)
glycosidic bonds, which are found in the structural polysac-
charide CGN (Lemoine et al., 2009). a-Amylase can also be
found in the intestinal bacteria of the colon (Ramsay et al.,
2006). However, as amylase cannot recognize the unique
glycosidic bonds in CGN, it is not involved with its
degradation. Galactosidases (also known as glycoside hydro-
lases) are present in the small intestinal mucosa of the human
and rat. In humans, three enzymes have been identified in the
intestinal mucosa (George, 1971; Gray & Santiago, 1969).
Two of these enzymes have activity toward the disaccharide
sugar lactose (glucose–galactose). This bond is a b (1–44)
glycosidic linkage. Lactase is located along the brush border
membrane in the small intestine. Although this enzyme has an
affinity for lactose, it is also believed to be capable of
recognizing and cleaving other molecules with b (1–44)
bonds. CGN has a unique a (1–43) bond that alternates with
the recognized b (1–44) bond. Therefore, while CGN
cleavage at the a-bond may not occur; it may be possible to
Esophagus
(A) (B)
Stomach
Duodenum
Colon Ileum
Jejunum
RectumVermiformappendix
Cecum
Esophagus
StomachDuodenum
CecumColon
IleumRectum
Jejunum
RatHuman
Figure 3. Differences in the gastrointestinal tract of human and rat. In humans (A), most digestion and absorption occur in the duodenum and lowerjejunum. In contrast, the rat (B) gastrointestinal tract is primarily jejunum and digestion begins in the stomach and continues throughout thegastrointestinal tract. Source: DeSesso et al. (2001) Food & Chem Tox, 39:211.
villi
crypts
lamina propria
muscularis mucosae
Figure 4. Diagrammatic representation of intestinal villi and cryptregions. Left: longitudinal section. Right: cross section viewed for eacharea. Microvilli increase intestinal surface area, which improvesabsorption of food nutrients. These cells are in constant flux movingfrom the crypt to the villi where they slough off into the intestine.Source: Creamer B. (1967) Br Med Bull 23(3):226–230.
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have cleavage at the b (1–44) bond (Bhattacharyya et al.,
2010a) provided the conformation of CGN allows recognition.
This has only been demonstrated under in vitro conditions
with purified enzymes. The existence and concentration of
enteric enzymes capable of degrading CGN are not known.
In addition, given the high molecular weight and the need
to penetrate bacterial cell walls in order to be subjected
to degradation, it is unlikely that this mechanism has
significance in vivo.
Breakdown products from in vitro incubation of CGN with
different galactosidases had differential effects on the release
of one proinflammatory cytokine, interleukin-8 (IL-8), in the
human colonic cell line (NCM460) (Bhattacharyya et al.,
2010a; Moyer et al., 1996). However, whether these break-
down products are formed in vivo in the GI tract is unknown
and many of the enzymes used in this study are either not
present or are found in low amounts in the human GI tract.
In addition, it is difficult to know exactly which enzymes were
Figure 5. Cell types and injury response ofthe intestinal tract. The lumen of the intes-tinal tract is lined by a mucosal layer andepithelial cells. On the basal side of thesecells, are the cells of the innate immunesystem. Macrophages, T-cells, and B-cellsreact to chemical or disease-relatedinjury to the intestinal epithelium.Source: Brenchley et al. (2008) MucosalImmun 1(1):25.
Epithelial cell
Secretory IgA
Neutrophil
Commensal bacterium
M cell
Gut lumen(A)
(8)
(3)
(2)
(4) (5)
(6)
(7)
(1)
(B)
CD4 T cell
CD8 T cell
B cell
Macrophage
Defensin
Bacteria
Infected CD4 T cell
Dendritic Cell
HEV
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used because proper (EC or enzyme commission classifica-
tion) nomenclature was not provided. In this study, the
enzymes used for CGN digestion experiments in vitro
included several recombinant a-galactosidases prepared
from Escherichia coli and purchased from a commercial
supplier. This enzyme recognizes and cleaves a-galactosyl
moieties from glycolipids and glycoproteins. These molecules
often derived from plant material are also called galactooli-
gosaccharides (GO). Examples of GOs include melibiose and
raffinose, both of which are found in soy products.
a-Galactosidases aid in the digestion of these plant-derived
molecules, which can be present in human diet. The enzyme
can be found in high levels in the fungi Aspergillus terreus
and Penicillium griseoroseum (Falkoski et al., 2006;
Ribon et al., 2002).
Humans and monogastric animals lack the enzyme
a-galactosidase (EC 3.2.1.22) (Falkoski et al., 2006). Most
of the gut microflora in humans is composed of bacteria
and although some fungus species undoubtedly exist, little is
known about them. However, efficient breakdown of GOs by
galactosidases requires dietary supplementation. Therefore,
these enzymes would be expected to have little relevance
to the intestinal breakdown of CGN because they are either
absent or in very low concentration. Carrageenase is an
enzyme that can break down CGN; however, this enzyme
is only found in marine organisms and has not been reported
to be present the intestine of humans or rodents (Lemoine
et al., 2009). Most importantly, there is no scientific evidence
for enzymatic degradation of CGN to poligeenan
(Mw 10 000–20 000 Da) by intestinal enzymes or by enzymes
of intestinal microflora.
Under harsh in vitro conditions, acid hydrolysis of CGN
at a pH of 3 and a temperature of 100 �C for more than 40 h
are required to obtain only a small fraction of hydrolytic
products (Rochas & Heyraud, 1981) which was obtained
using gel chromatography and changes in viscosity (Figure 6).
It has been suggested that CGN can undergo acid hydrolysis,
in the stomach of rodents and humans, to poligeenan with
an average molecular weight 520 000 Da. This conversion
has not been demonstrated in animals or humans.
Furthermore, it is chemically improbable to have any
significant acid hydrolysis in rodents based on the higher
pH values (3–5) of rodent stomachs, the less than optimal
temperature of the body (37 �C) for acid hydrolysis of CGN,
and the short (3–4 h) residence time in the stomach. There
will be more discussion on this later.
Early in vitro work by Ekstrom (Ekstrom, 1985; Ekstrom
et al., 1983) is often cited as supporting evidence for acid
hydrolysis of CGN to poligeenan in animals. These studies
reported that k-CGN was more susceptible to degradation
than i-CGN in simulated gastric juice at pH 1.2 and 1.9, with
considerable degradation of k-CGN after a 4 h incubation in
simulated gastric juice (containing 0.1 M LiCl and 0.1 N HCl
at pH 1.2). At first glance, these findings seem contradictory
to the discussions above; however, there are four important
caveats to this work: (1) the experiments were done in the
absence of protein or a food matrix. The absence of protein
would increase the portion of free CGN available for
hydrolysis and hence exposure to intestinal cells. In compari-
son, the presence of food protein would bind CGN making it
significantly less available for hydrolysis and reduce cellular
exposure. (2) LiCl was added to the CGN preparation, which
would deconform the CGN helical structure and increase the
efficiency of hydrolysis, a situation not found in animals or
humans (Capron et al., 1996). (3) The pH of the stomach of
rats is maintained at a pH between 3 and 5 (DeSesso &
Jacobson, 2001) and the pH in the human stomach following a
meal is between 4 and 5 and then decreases as the food leaves
the stomach (Kararli, 1995). And (4) animal studies in which
CGN or poligeenan were administered in drinking water
should be viewed differently because CGN in drinking water
has limited bonding with protein and, therefore, would be
more available to hydrolysis. Most commercial uses of CGN
intended for human exposure are formulated into food
products where the CGN is bound to protein. There is no
evidence that poligeenan is formed in the GI tract of either
rodents or humans when CGN is bound to protein. Based on
the preceding experimental information, it is incorrect to
suggest that gastric acid hydrolysis of food-grade CGN bound
to protein occurs in rodents or in humans.
The in vitro data discussed above are supported by a series
of animal studies in which rats were administered poligeenan
or CGN in their diet. Animals that received poligeenan
showed the greatest response with sustained increases in
cellular proliferation and cell transformations associated with
the development of intestinal ulcers or tumors (Ishioka et al.,
1985, 1987; Oohashi et al., 1981; Wilcox et al., 1992).
Such changes were not seen in animals administered CGN in
the diet (Weiner, Part II, 2014). Some researchers have
intimated that CGN is susceptible to hydrolysis by the low pH
of the stomach and that it can be degraded by microflora in
the colon (Tobacman, 2001). This concept was refuted by an
International Program on Chemical Safety (IPCS WHO Series
f ≤ 40 h
f = 55 h
f = 75 h
f = 148 h
VT VO
D
C
B
A
Figure 6. Carrageenan resistance to acid hydrolysis. Elution profiles ofcarrageenan treated with sulfuric acid (pH 3.0) at 100 �C for varioustimes with stirring. Following the incubation period, the samples wereneutralized and prepared for chromatography (Rochas & Heyraud, 1981).Size separation was done by gel chromatography. High molecular weightcomponents elute in the void volume (Vo), smaller fragments ofhydrolysis elute in subsequent elution fractions (D, C, and B). FractionC is D-galactose as determined by Infrared and NMR spectroscopies andelutes near the total volume of elution (Vt). Source: Rochas and Heyraud,1981.
8 J. M. McKim Crit Rev Toxicol, Early Online: 1–33
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42, Food Additive series, 1999) in which reviewers did not
consider degradation of food-grade CGN in the gut to be
toxicologically significant. Thus, although CGN can undergo
acid hydrolysis in the laboratory when subjected to acid at
low pH (1–3) under high temperature (80–100 �C) for
extended periods of time (440 h) to form poligeenan; these
conditions do not occur in animals or in humans.
It has also been suggested that CGN can be degraded by
intestinal microflora to form poligeenan. This process is
highly unlikely in rats or humans as CGN is considered to be
an indigestible polysaccharide (Ikegami et al., 1990). CGN is
inert to enzymatic hydrolysis by intestinal secretions in both
human and monogastric animals (Harmuth-Hoene &
Schwerdtfeger, 1979). In addition, intestinal degradation is
disputed because CGN is foreign to most human gut flora and
enzymes, and the molecular weight profiling of CGN in the
diet and feces showed limited cleavage (Uno et al., 2001).
This information is important because many of the early
animal studies assumed that CGN could be degraded (dCGN)
in the GI tract to poligeenan and as a result the term CGN was
improperly used interchangeably with dCGN. In fact, many
articles referenced in more recent studies cite these early
works by stating that CGN causes GI toxicity and that CGN
has been used in multiple studies to induce GI ulcers or to
produce a model of irritable bowel syndrome (Borthakur
et al., 2007; Tobacman, 2001). If the articles cited in the
introduction of the Borthakur paper are reviewed, it is clear
that these referenced studies were actually conducted by
dosing animals with poligeenan (dCGN) and not the food
additive CGN (Marcus et al., 1992; Moyana & Lalonde, 1990;
Onderdonk et al., 1985).
In vitro studies and their relevance to human hazardand risk assessment
Confusion regarding CGN nomenclature and the differences
between CGN and poligeenan has resulted in incorrect
conclusions regarding food-grade CGN and in vivo effects.
This, in turn, has led to the incorrect use of the in vivo
findings to support mechanism-based hypotheses to explain
and support in vitro or cell-based studies. The result has been
a plethora of in vitro studies using human intestinal cell lines
(NCM460), human hepatoma cells (HepG2), human mono-
cytic cells, and human breast cells grown in culture all
designed to identify intracellular mechanisms that could
explain the in vivo findings. These in vitro studies were
intended to identify the underlying mechanisms of intestinal
inflammation and ulcers that were reported in early animal
studies and incorrectly attributed to food-grade CGN. As
discussed above, these in vivo effects were due to the
administration of poligeenan.
Many of the in vitro or cell-based studies have suggested
that CGN can bind to and activate several membrane receptor
signaling pathways that are involved with inflammation,
diabetes, breast cancer, and the innate immune system.
Because the early animal findings combined with the newer
in vitro data are being used incorrectly to try to support
potential adverse effects of food-grade CGN, it is important to
review the published data collected from these studies and
compare these findings to adverse events measured in
animals. The intent is not to demonstrate that the data
collected from the in vitro studies are incorrect, but rather to
discuss their relevance to hazard identification and risk
assessment to humans who ingest food-grade CGN as it is
used as a food additive.
Ideally, in vitro data should only be used to identify hazard
and predict health risk when the following conditions exist:
(1) the test chemical has been well characterized in terms of
identity and purity, (2) the proposed pathway of the adverse
effect implicated in vivo is present and functional in the
in vitro system, (3) the test chemical can and does interact
with the putative pathway receptors, (4) the in vitro system
has been shown to be a good surrogate model for the in vivo
effect, and (5) the known or suspected pathway in vitro is
linked biologically to an in vivo event. For example, the
abundance of the TLR4 receptor varies throughout the GI
tract (Ishihara et al. 2006) and protective mechanisms in the
GI tract that prevents lipopolysaccharides (LPS)-mediated
activation are not present in liver. The liver expresses TLR4
receptors and responds to LPS, however, some liver tumor
cell lines (e.g. HepG2) do not possess high levels of TLR4
(Nishimura & Naito, 2005). Thus, there are examples of
receptors that vary in tissue distribution in vivo and in vitro,
and can have different sensitivities to ligands in different parts
of the body (Ishihara et al., 2006).
It is not enough that the cell model comes from the correct
tissue, it must also express the receptor of interest and that
receptor must have the same function as its counterpart
in vivo. The in vitro model should represent the correct target
species. For example, using a dog hepatocyte to study human
liver toxicity may not provide an accurate interpretation due
to biochemical differences between dogs and humans.
Observing an adverse effect in an animal model does not
necessarily mean that the same effect will occur in humans.
Species differences should be incorporated into the analysis of
data obtained from both the in vitro and the in vivo models.
The physical and chemical properties of the test compound
(e.g. purity, stability, solubility, partition coefficients, and
protein binding) must be understood and accounted for in the
data analysis, hazard identification, and risk assessment
process.
An example of in vitro versus in vivo mechanisms can
be seen with glucose metabolism. Glucose uptake by cells
in animals and humans requires insulin, and both insulin
receptors and glucose transport proteins must be present
and functional on the cell surface. Most tumor cell lines,
such as the human hepatoma (HepG2) cell line do not
require insulin to grow, yet glucose is present in excess
amounts in standard culture medium. In comparison,
primary hepatocytes in culture do require insulin in
order to survive. Thus, using a tumor cell line to study
insulin-mediated glucose metabolism would not be appro-
priate since the primary mechanism of glucose uptake is
not present or not functioning with the same importance
as it does in vivo. Thus, when in vitro cells do not
function in a way that mimics normal human organ or
tissue biology in vivo, the in vitro model cannot be used
to identify chemical perturbation of that system.
If cells are being used to assess chemical interaction with
membrane surface receptors, the binding and activation
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should be confirmed with purified receptor. Cell culture is a
static system; large molecules can coat cells in culture and,
in so doing, can either inhibit or activate many membrane
surface receptors in an indirect or a non-specific manner.
Again, this would not be the case for tissues in vivo, which are
bathed in plasma, a dynamic system, or for compounds that
bind to food proteins and are subject to the motility of the GI
tract. Surface receptors are generally highly specific for their
substrates because activation can initiate significant bio-
logical events. Molecules that appear to bind to many
different receptors should be viewed with skepticism until a
direct binding association can be determined. Clearly, there
are many factors that dictate the use of in vitro data for
predicting in vivo events in animals or humans.
In order for in vitro studies to have relevance to – or be
predictive of – events measured in animal studies, the in vitro
models should reflect mechanisms related to the biological
effects observed in vivo. Changes to cellular biology
measured using in vitro cell-based systems may not be
relevant if the events in vitro cannot be linked to observations
in vivo. The biological effect in vivo that is most likely to
occur for insoluble and indigestible food additives, such as
CGN, is induced proliferation of the cells in the GI tract;
however, this was not shown in dietary studies in which high
levels of food-grade CGN were used (Cohen & Ito, 2002;
Weiner et al., 2007; see Weiner Part II, 2013).
CGN and effects on the GI tract of animals
Some studies in which CGN was administered to rodents
reported proliferative responses in the GI tract that included
hyperplasia and hypertrophy of the small intestine. These
effects were more pronounced and lasted longer after
cessation of treatment when poligeenan was administered
(Calvert & Reicks, 1988; Calvert & Satchithanandam, 1992;
Wilcox et al., 1992). The authors concluded that these
observations were adverse and a direct consequence of CGN
binding to or reacting with intestinal mucosa. However,
another interpretation was provided by Ikegami et al. (1990).
This study compared the effects of several indigestible
polysaccharides, which included pectin, CGN, locust bean
gum, xanthan gum, and guar gum in the diet of rats at high
doses (45%). The results showed an increase in the size of the
small intestine, cecum, and large intestine or rats (Ikegami
et al., 1990). These effects were viewed by the authors as
adaptive in response to indigestible viscous agents in the GI
tract. The presence of these agents reduced the absorption of
food, which caused an increase in the activity of exocrine and
pancreatic–biliary function as the body attempted to improve
the efficiency of digestion. This, in turn, led to hyperplasia
and hypertrophy of the digestive organs and increased
secretion of digestive juices (Ikegami et al., 1990; Poksay &
Schneeman, 1983). These indirect physiological effects are
compensatory and transient in nature and should not be
considered adverse events.
Three studies, in which various forms of CGN were
administered to Fischer 344 rats in diet, have been reported
from two different laboratories: Proctor and Gamble
(Cincinnati, OH), and the Experimental Nutrition Branch or
the US FDA (Calvert & Reicks, 1988; Calvert &
Satchithanandam, 1992; Wilcox et al., 1992). These studies
were well designed and included proper controls. The test
groups consisted of CGN, poligeenan, and guar gum. The
route of administration was dietary and the duration was for
28 and 90 d. The 90-d Proctor and Gamble study (Wilcox
et al., 1992) included a 28-d recovery group. The purity of the
CGN could not be determined. All three studies used the
same biomarker for cell proliferation (TK activity) and some
included histological examination of the intestine. In the two
FDA studies (Calvert & Reicks, 1988; Calvert &
Satchithanandam, 1992), there was a statistically significant
increase in TK activity following CGN treatment, but only at
the highest exposure. There were no histological changes
noted at any dosage, and there was no observable change in
mucin histochemistry. CGN and poligeenan administered in
the diet of Fischer 344 rats caused a five-fold increase in TK
activity in both the CGN and poligeenan groups and the
number of proliferating cells in the upper third of the crypt
increased 35-fold. However, following a 28-d recovery
period, TK activity of the CGN-treated animals returned to
basal levels, but animals treated with poligeenan had activity
levels that remained elevated by two-fold relative to control
animals. In a fourth study by Weiner et al. (2007), Fischer
344 rats were exposed in the diet for 90 d to 0, 2.5, and 5%
k-CGN, which was well characterized in terms of its purity
and molecular weight. This study did not measure TK
activity in the intestine, but did perform extensive histo-
logical analysis of multiple organs, particularly the GI tract.
This latter study is in agreement with the studies done earlier
with regard to animal health and histological changes in the
GI tract. This study was also conducted in compliance with
GLP. In conclusion, CGN did not result in any histopatho-
logical changes at any level of CGN feeding (Calvert &
Satchihanandam, 1992). In all three studies, CGN in the diet
did increase cell proliferation in the colon, as measured by
TK activity. This effect was more pronounced in animals
treated with poligeenan and upon cessation of treatment,
CGN animals returned to normal levels while dCGN animals
had TK activity that remained two-fold above controls
after the recovery period (Wilcox et al., 1992). Colon
cancer is associated with an increased number of crypt
cells in S-phase. Although TK activity can be an indicator of
cell proliferation, it does not provide a direct measure of an
increased ratio of cells in S-phase, an important indication of
cancer. TK activity increases in 26% of human solid tumors
and, in this subset of cells, TK activity is associated with a
high ratio of cells in S-phase (Keri et al., 1988). In the
absence of tumor formation, TK activity should be normal-
ized to cells, not to protein, in order to demonstrate that
increases in activity are correlated with increased cell number
(Cohen & Ito, 2002). The most accurate method for
determining the ratio of cells in S-phase is immunohisto-
chemical staining and detection of proliferating cell nuclear
antigen (PCNA) performed on colon tissue sections. In the
studies reported by Calvert & Reicks (1988), Calvert &
Satchithanandam (1992) and Wilcox et al. (1992), these
techniques were not used. As a result, Cohen & Ito (2002)
conclude that it is highly unlikely that CGN is increasing cell
proliferation of the critical crypt stem cells, which is
necessary for colonic carcinogenesis.
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An important question to answer is whether or not the
increased cell proliferation was due to an adaptive response of
the GI tract to insoluble and indigestible material (e.g. CGN,
cellulose, pectin, guar gum) or due to a specific chemical
interaction of CGN with cells. In two studies in which
several bulking agents were administered to rats, it was
concluded that an adaptive response to high levels of
indigestible agents can indeed occur (Ikegami et al., 1990;
Poksay & Schneeman, 1983).
Effects of CGN on glucose metabolism
Glucose is the precursor molecule for the production of all
cellular energy through anaerobic (glycolysis) and aerobic
(oxidative phosphorylation) processes. The primary tissues
involved with glucose uptake from blood are muscle, liver,
and fat. Glucose uptake is by facilitative diffusion. The uptake
of glucose from blood requires a coordinated and complex
signaling process that involves insulin, phosphotidyl inositol
triphosphate (PIP3), several kinases, and glucose transporters
2 and 4 (GLUT2 and GLUT4) (Figure 7).
The signaling process begins, in skeletal muscle or fat
tissues, with the binding of insulin to the insulin receptor
located on the plasma membrane. This activates a series of
protein kinases leading to the activation of PKB/Akt, the final
trigger for signaling GLUT4 receptors to move to the plasma
membrane where they bind glucose and transport it into the
cell. Chemical disruption of this process can occur by
inhibiting insulin or glucose binding to their respective
receptors or by chemical interference with the complex array
of intracellular signaling processes (Paul et al., 2007).
Bhattacharyya et al. (2012) reported that C57/BL6J mice
exposed to �–k–CGN mixture in drinking water at a dose of
10 mg/L for 18 d had an impaired ability to clear glucose from
the blood as determined by a glucose tolerance test (GTT).
There was a statistically significant lag in the rate of glucose
uptake, relative to control animals. An insulin tolerance test
(ITT) was done on animals following exposure to �–k-CGN
for 33 d. This test showed that CGN-treated animals had a
statistically significant resistance to insulin.
Based on estimates of daily water consumption by mice
of 6 mL/day (Bachmanov et al., 2002), the estimated daily
dosage would have been 10 mg/mL� 6 mL¼ 60 mg/d. If this is
corrected for average body weight of a mouse of 30 g
(0.03 kg), the daily dosage was approximately 2000 mg/kg/d or
2.0 mg/kg/d� 18 d of exposure provides a total intake of
36 mg. The Scientific Committee of Food in Europe allows an
average daily intake of 75 mg/kg/d (SCF, 2003). It has been
estimated that the average human daily intake of CGN in food
is about 40 mg/kg/d (JECFA, 2008). Thus, the study by
Bhattacharyya et al. (2012) provided a dosage in drinking
water that was below the average daily intake allowed for
humans. Although the dosage of CGN used in this study
appears relevant to human exposure scenarios, the form of
CGN and the formulation of administration are quite different.
The primary use of CGN in food is stabilization and
thickening. The �–k-CGN mix used in the in vitro study
discussed is composed of CGN types that do not form the
most stable complex molecular structures observed when
bound to food proteins, and placed into aqueous solution.
In water, CGN does not form a helical (non-gelling) structure
and, as such, is more available and susceptible to breakdown.
This point is important because it does not negate the in vitro
observation reported, but it does lessen its significance with
regard to human hazard and risk assessment. This important
issue of in vitro concentrations translated to in vivo dosage is
important and is addressed in more detail in the accompany-
ing review Part II by Weiner (2013).
Because insulin resistance can cause glucose intolerance,
the study evaluated the effects of CGN on various components
of the insulin signaling pathways. These experiments showed
that in the liver of mice exposed to CGN in drinking water,
and in a human hepatoma cell line (HepG2), there was a
reduction in the amount of phosphorylated protein kinase B
P
Insulinreceptor
Insulin
IRS-1/2
PI-3K
PI-3K
IRS-1
/2
PIP2
PIP3PIP3
PIP3
PTEN
PDK-1
PKC?ζ
Thr308Ser473 PKB/Akt
GLU
T4
GLU
T4
Glucose
GLU
T4
Figure 7. Complex signaling required for glucose uptake from blood into cells. Uptake of glucose from blood occurs primarily in muscle, fat, and livertissues. The glucose receptors required for uptake as well as the role of insulin signaling vary between liver and muscle. In muscle or fat, facilitateduptake of glucose from blood is accomplished by insulin-stimulated recruitment of GLUT4 to the plasma membrane. In the liver, insulin signals theinhibition of glucose storage pathways and buy has less of an effect on GLUT4 as GLUT2 is the primary hepatic glucose transporter. Source: Paul et al.(2007) Environ Mlth Persp, 115:740.
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(Akt), without a reduction in the absolute amount of Akt
protein. The authors put forth a proposed mechanism to
explain these findings that hypothesize that CGN adminis-
tered orally is absorbed from the gut, enters the liver, binds
specifically to and activates toll-like receptor 4 (TLR4)
signaling through B-cell CLL/lymphoma10 (BCL10), which
increases phosphorylation of the protein called inhibitor of
nuclear factor kappa-B kinase subunit beta (IKK-b). IKK-bthen stimulates phosphorylation of insulin receptor substrate
1(IRS-1), which inhibits insulin-mediated signaling and
subsequent recruitment of GLUT4 to the plasma membrane,
thereby reducing glucose uptake into the liver (Bhattacharyya
et al., 2012).
It is important to remember that although the liver is a key
organ for glucose metabolism, the role of insulin and the
signaling processes that control glucose uptake in liver are
considerably different from those in skeletal muscle and fat
tissue, which are the primary sites of glucose removal from
blood in the insulin-stimulated state in vivo (DeFronzo et al.,
1992; Kim et al., 1999). In the liver, insulin can activate Akt,
which in turn, activates glycogen synthase kinase, a signal for
the liver to increase glucose by inhibiting its conversion to
glycogen. Unlike skeletal muscle and fat tissue, glucose
uptake from blood into liver is accomplished by facilitative
transport mediated by GLUT2.
GLUT2 is always present on the plasma membrane of the
liver, and therefore, glucose uptake can occur independently
of insulin. The increase in intracellular glucose induces
GLUT2 mRNA production, causing an increase in GLUT2
transporter protein and an increase in glucose influx (Osawa
et al., 2011; Rencurel et al., 1996). It is clear that although the
role of insulin activation of Akt represents an important
signaling event for glucose uptake, the functional role of
insulin and downstream targets, such as Akt, varies with
tissue type. With these mechanistic points in mind, data
collected using a human hepatoma cell line should be viewed
cautiously when attempting to extrapolate these in vitro
observations to in vivo events.
Although mice exposed to CGN showed a reduced rate of
glucose uptake, overall glucose levels in the mouse under the
conditions tested by Bhattacharyya et al. (2012) seem to fall
within normal ranges reported by others (Andrikopoulos
et al., 2008) (Figure 8A and B).
The graphs in Figures 8(A) and (B) were taken from
Andrikopoulos et al. (2008) and from Bhattacharyya et al.
(2012). Note the similarity in the glucose levels.
Both studies were done using C57BL/6J mice, both received
glucose via intraperatoneal injection. The mice used to
generate the data in graph on the left (Figure 8A) were
fasted for 6 h, while those in the graph on the right
(Figure 8B) were fasted for 18 h. The data in Figure 8(A)
shows mice fed a normal diet (solid squares) versus mice
fed a high fat diet (open squares). The graph shown in
Figure 8(B) depicts mice fed a normal diet without CGN in
the drinking water (lower curve) compared to mice that
had been treated for 18 d with (10 mg/L) CGN in their
drinking water (upper curve). This route of exposure could
be problematic because �-CGN in water is always in the
disorganized or non-gelling conformation, which in the
absence of protein, can facilitate more direct contact with
cells, a situation that would not be expected to occur when
CGN is administered in diet (Weiner Part II, 2013;
Blakemore & Harpell, 2010). Thus, although CGN in
drinking water produced a lag in glucose uptake and insulin
resistance in mice, the absolute levels of glucose remain
within a normal range. While interesting, these data should
not be used to establish hazard or assess risk related to CGN
in humans who are exposed to CGN in diet.
It is generally believed that when CGN is administered
orally that the majority of it remains intact as it passes
through the gut because it can be quantitatively recovered in
the feces (Arakawa et al., 1988; Uno et al., 2001; Part II
Weiner, 2013). These studies were done with CGN mixed
with diet and hence CGN was bond to food proteins. In recent
in vivo studies Bhattacharyya et al. (2012), CGN was
administered in drinking water, which can make it more
available for hydrolysis (see Part II Weiner, 2013 for further
discussion).
In contrast, humans are exposed to CGN in food and CGN
binding with food proteins is known to be high (Blakemore &
Harpell, 2010). As food enters the stomach gastric pH
increases. There is an absence of microflora in the upper GI
tract, and rapid transit time, all of which would work together
to reduce cellular exposure to CGN or its putative breakdown
products in these regions of the GI tract. If one accepts the
general view that high molecular weight CGN cannot be
readily absorbed and that little degradation of CGN occurs in
the gut, then any systemic effects reported following the oral
dietary administration of these compounds must be viewed
cautiously. Direct compound effects are unlikely; however,
this does not rule out the possibility of indirect effects, such as
osmotic or changes in cation flux.
Figure 8. A comparison of glucose tolerancetests performed in mice. (A) Data fromAndrikopoulos et al. (2008) (under leftfigure). (B) Data from Bhattacharyya et al.(2012) (under right figure). The lag time forglucose uptake in mice is compared betweentwo similar studies. The plasma glucoselevels in both studies are similar and withinnormal range. IPGTT denotes an intraperito-neal injection of glucose (A). The opensquares indicate high fat diet, the solidsquares normal diet (A). Mice exposed toCGN in drinking water upper curve versuscontrols lower curve (B). It is important tonote that the identity and purity of the CGNused in panel B was not clearly reported.
50.0
40.0
30.0
20.0
10.0
0.00 30 60 90 120
Pla
sma
gluc
ose
(mm
ol/L
)
Time (min)
0 15 30 60 90
Time (min)
IPGTT
*
#
35
30
25
20
15
10
5
0
Glu
cose
(m
mol
/L)
(A) (B)
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In vitro studies using cell lines must make every attempt to
mimic as closely as possible the formulation of the test
material, and the concentrations expected in humans. Thus,
in vitro experiments that attempt to demonstrate whether
CGN could cause direct effects on the GI tract or indirect
effects to systemic organs must be taken into account and
incorporate the unique chemical properties of the CGN
molecule in order for the in vitro data to be extrapolated to
in vivo mechanisms. Thus, although it is possible that CGN
could cause direct effects on the GI tract or indirect effects to
systemic organs, experiments in vitro that attempt to show this
must understand and incorporate the unique chemical proper-
ties of CGN in order for the in vitro data to be extrapolated
to in vivo mechanisms. A fundamental component to in vitro
experiments is to demonstrate that a test material can cross
biological membranes, this information confirms cellular
uptake and can verify the active agent producing the
observed changes.
The exposure of mice to CGN in drinking water and the
subsequent effects on glucose uptake and insulin sensitivity
are interesting; however, it would be helpful for the
interpretation of this work, if the amount of CGN and the
form (intact, degraded) actually absorbed had been measured.
In addition, detailed information regarding the binding of
CGN and poligeenan to the insulin receptor is needed in order
to understand the mechanism and affinity of binding. Most of
the biochemical effects reported have identified membrane
receptors as the initiating target. This includes the TLR4 and
the insulin receptor. Many of these studies were conducted
using cell models, and it is unclear whether the effects
measured were due to direct interaction between CGN and the
putative receptors, or simply an artifact of the experiment
caused by a high molecular weight polymer (Mw¼ 200 000–
800 000 Da) coating the cell surface.
Diabetes has been linked to activation of TLR4 signaling
pathways in adipocytes, macrophages, and bone marrow
(Mohammad et al., 2006; Shi et al., 2006; Song et al., 2006),
but a connection between TLR4 activation and diabetes
mediated by inflammatory pathways in the GI tract is less
clear. TLR4 is down-regulated and under tight negative
control in the gut, which protects the host from exposure to
endotoxin produced by normal and required gut microflora.
Bhattacharyya et al. (2012) report that mice exposed to
(10 mg/L) CGN in drinking water showed a reduction in
glucose uptake and alternations in insulin sensitivity and
signaling. The premise for this work was that CGN binds to
and activates TLR4 in the GI tract, which in turn activates
NFkB and the inflammatory response (Bhattacharyya et al.,
2008b,c, 2010a,b, 2011; Borthakur et al., 2007). Because
TLR4 expression and activity are extremely low in the GI
tract and because TLR4 is not as sensitive to LPS in the
intestine, due to decoy molecules designed to protect the
intestinal environment from LPS induced inflammation, it is
possible that TLR4 in the gut is less important in terms of an
LPS-mediated inflammatory response. Although detailed
mechanistic data indicating a CGN-mediated activation of
TLR4 signaling pathways in the human intestinal epithelial
cell line (NCM460) has been reported by this group, the cell
line used as the test system may not accurately mimic TLR4
function and expression in the rodent or human intestinal EC.
Cell models can be useful tools for identifying potential
effects of chemicals on signaling pathways that may lead to an
adverse outcome in animals or humans. However, the use of
these systems to investigate potential effects of CGN on key
signaling pathways requires special knowledge of CGN
chemistry, protein binding, purity, and solubility. Moreover,
it is also important to understand how cell models are similar,
as well as dissimilar, to the in vivo situation. In many
instances, the function and activity of key signaling pathways
in animal and human tissues behave much differently than
they do in cell lines. For example, HepG2 cells have been
reported to have no TLR4 (Liang et al., 2011; Nishimura &
Naito, 2005; Wei et al., 2008) and be insensitive to the
cytotoxic effects of the proinflammatory cytokine, TNF-a,
while other studies have shown that HepG2 cells express low
to high levels of TLR4 mRNA and protein (Hsiao et al., 2013;
Xia et al., 2008). It is also important to note that HepG2 cells
grown in culture medium with glucose revert to glycolysis
(CrabTree Effect) for energy. However, if the glucose is
replaced with galactose the cells maintain oxidative metab-
olism and are sensitive to mitochondrial effects (Marroquin
et al., 2007; Schoonen et al., 2012). The source of cells
(animal or human), normal or tumor, and culture conditions
used can significantly influence experimental results. Hepatic
TLR4 substrates include LPS, lipoteicholic acid (LTA), and
Taxol. Binding of these substrates to the TLR4 receptor
initiates induction of intracellular pathways linked to inflam-
mation (Takeuchi & Akira, 2001). The in vitro work reported
demonstrates that there is interaction with TLR4, but without
the supporting information of cell culture conditions, protein
binding and the purity of the test material it is not possible to
ascertain whether the observed effects are due to CGN or to
contaminants present in the CGN preparation or in the cell
culture system. Ideally, the expression (mRNA) as well as
protein levels of TLR proteins should be assessed in any cell
model employed to ensure that the pathway being measured is
present and functional. HepG2 cells do not require insulin to
grow, or to take up glucose from the medium, a situation
shared by most tumor cell lines. As a result, the necessary
signaling pathway for glucose uptake in the HepG2 cells is
disrupted, and not indicative of the liver in vivo. Because the
cells used in the study by Bhattacharyya et al. (2012) were not
cultured with galactose, it is possible that they may have been
more sensitive to glucose uptake inhibition. Moreover, for
some experiments serum was removed from the media. Serum
withdrawal from these cells is known to induce apoptosis, so
culturing these cells in serum-free medium would initiate
these events (Bai & Cederbaum, 2006).
When data describing adverse events in rodents or in cell
models are intended for safety assessment in humans, the test
systems must be shown to possess the same functional
mechanisms as humans, a clear dose and time effect should be
demonstrated, and dose, formulation, and route should mimic
human exposure pathways. The purity and composition of the
CGN used in several of the studies discussed in this review
were not reported. Although the amount of CGN administered
to animals and exposed to cells in vitro is reportedly less than
the amounts predicted for human daily intake, it is unclear
how the low exposure levels used in vitro relate to average
daily exposure in humans. Moreover, it is important to
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administer test materials to cell or animal test systems in the
same form to which humans would be exposed. The amount
of any test material that is administered orally does not equate
to the systemic or absorbed dose. Given that most drugs,
which are intended to be absorbed from the gut, have less than
100% bioavailability and much lower molecular weights (for
conventional drugs, 200–600 Da) (Schulz & Schmoldt, 2003;
Veber et al., 2002), it is likely that the actual systemic
exposure to CGN (bioavailability of CGN, poligeenan, or low
molecular weight components all of which have much higher
molecular weights than drugs) is extremely low and far below
the administered dosage. This assumption is supported by
several animal studies that reported that CGN could be
quantitatively recovered from feces (Uno et al., 2001).
In vitro studies and CGN effects on cell signalingpathways
Effects of CGN on Wnt and bone morphogeneticprotein (BMP) signaling pathways in NCM460 cells
Wnts make up a large family of secreted proteins character-
ized by the presence of a lipid modification (palmitoylation)
on one of the cysteine residues. Wnt proteins vary between
350 and 400 amino acids in length, possess 22–24 conserved
cysteines, are highly hydrophobic, and show 20–85% amino
acid identity within the Wnt family. Wnt signaling proteins
bind to a cell surface receptor protein known as Frizzeled
(FRZ). This activates another family of receptors called
Dishevelled (DSH). The Wnt–FRZ–DSH complex binds to
a low-density lipoprotein receptor-related protein (LRP)
(Figure 9). This complex of Wnt protein bound to the
multiple receptor complex results in an accumulation of a
protein known as b-catenin, which can translocate to the
nucleus and bind to TCF/LEF-1 transcription factors, and
promote the expression of specific genes related to cell health.
The binding and subsequent activation of DSH is an
important step in this cascade. When activated DSH inhibits
a second complex of proteins, composed of axin, glycogen
synthase kinase 3 (GSK-3) and protein APC, the axin–GSK–
APC complex facilitates the degradation of b-catenin thereby
providing a negative control mechanism for gene expression
(Figure 9). Thus, when Wnt binds to and activates the
receptor complex, Axin is removed from the destruction
complex, which stabilizes cytoplasmic b-catenin allowing
some to enter the nucleus. Wnt proteins play important roles
in embryonic development, cell differentiation, pattern for-
mation, cell fate decision, axon guidance, and tumor forma-
tion. Alterations of Wnt proteins, adenomatous polyposis coli
(APC) protein, axin, and T-cell factors (TCF) have been
associated with carcinogenesis (Farrall et al., 2012;
Gregorieff & Clevers, 2005).
Another important signaling pathway in the GI tract is the
BMP pathway. BMP is a member of the transforming growth
factor-b (TGF) family of proteins. The maintenance of
intestinal homeostasis is dependent on cross-talk between
the Wnt and BMP signaling pathways. Wnt signaling
promotes the proliferation of Crypt progenitor cells and
migration from the Crypt to the villi. BMP controls cell
proliferation and differentiation by exerting control effects on
b-catenin, which is controlled by Wnt signaling mechanisms
(Figure 10). The BMP signaling pathway maintains intestinal
balance at multiple levels of tissue. At the epithelium, it
controls proliferation, in the mesenchyme, it controls
myofibroblasts, and local immune cells (Gregorieff et al.,
2012; Shroyer & Wong, 2007) (Figures 10 and 11).
CGN was evaluated in vitro for its effect on BMP and Wnt
proteins and its ability to activate the Wnt signaling pathway
(Bhattacharyya et al., 2007). In this study, a normal human
intestinal epithelial cell line (NCM460) was used as the test
system. The cells were exposed to either �-CGN (1 mg/mL),
k-CGN (3 mg/mL), degraded k-CGN (3 mg/mL; K4¼ 4
disaccharides; K14¼ 14 disaccharides), dextran sodium sul-
fate (DS; 0.8 mg/mL), or chondroitin sulfate (CS, concentra-
tion not provided) for 1–8 d. The culture media and cells were
collected at specified time points for analysis of cytokines.
Cytokine levels in the cell were done using an antibody
cytokine array, while excreted BMP4 protein in media was
measured by ELISA. BMP4 gene expression in cells was
measured by quantitative reverse transcription polymerase
chain reaction (qRT-PCR). The expression of Wnt mRNA was
measured using a Wnt super array containing 113 genes
related to Wnt signaling. The primary effector protein of the
Wnt pathway, b-catenin was measured using an ELISA. There
was activation of the Wnt pathway by CGN as determined by
Axin
receptorcomplex extracellular
“destructioncomplex”
cytoplasm
nucleus
CK1
APC GSK
P-
1
β-Cat
β-Cat
LEF
transcription
2
interaction
APC
AxinGSK P
P
β-Cat
cytoplasm
nucleus
translocation
proteolysis
ATP
ADPLRP
WNT
β-Cat
FRZ
DSHG
Figure 9. Components of the canonical Wnt signaling pathway. Wntproteins are highly conserved across species and are known to playimportant roles in developmental regulation including cell differenti-ation, polarity and oncogenic process. A primary downstream regulatorin the Wnt signaling pathway is b-cantenin (b-Cat). Wnt is a secretedlipid-modified protein. In order to initiate the canonical Wnt signalingpathway, Wnt must bind to a ligand which then binds to cell-surfacereceptors Frizzled (FRZ), the Wnt–FRZ complex then binds todisheveled (DSH) proteins, and then to lipoprotein receptor protein(LRP). This then signals the inhibition of b-Cat degradation, which iscontrolled by a second complex of proteins axin-glycogen synthasekinase (GSK)-adenomatous polyposis coli (APC) protein. This freesb-Cat to move into the nucleus where it controls gene expression atthe transcriptional level (Jansson et al., 2005; Kolligs et al., 2002;Sakanaka et al., 2000).
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an increase in b-catenin protein in the media. There was also a
reduction in the amount of BMP4 protein secreted and in
BMP4 gene expression following exposure to CGN. The data
for BMP4 protein levels following exposure to only �-CGN
and a CS control are shown in Figure 12(A–C).
In Figure 12(A), the effect of �-CGN (Cg) on BMP4
protein levels as a function of time was measured in spent
media with a BMP4-specific antibody and quantified by
densitometry. These data indicate that in the presence of
CGN, BMP4 excretion from cells is higher. In another
experiment, exposure of NCM460 cells to �-CGN (LCG),
k-CGN (KCG), low molecular weight fragments (K4 and
K14), DS, and CS, BMP4 was again measured in media by
ELISA, and normalized to cellular protein (Figure 12B).
These data indicate that LCG and DS, but none of the other
test groups, caused a depletion of BMP4 on both days 2 and 4
of treatment. This effect appeared to be independent of time
under the conditions tested. The lack of a time-dependent
response suggests that the BMP4 in media may not be
reflective of BMP4 in the intact cell or that the time course
for BMP4 secretion was shorter than 2 d. The authors state
that they checked BMP4 differences in cell lysate, but they
showed no data indicating the relative levels of BMP4 in cell
lysate versus medium, and it was not clear from the work
whether any statistical tests had been performed. DS has been
used to induce colitis in mice and rats. The DS-induced colitis
model is a well-established system used to test new drugs for
their effectiveness in the treatment of colitis. The molecular
weight of DS varies depending on the chain length, but the
form generally associated with colitis has a molecular weight
between 40 000 and 50 000 Da (Araki et al., 2006; Laroui
et al., 2012; Marrero et al., 2000). The source of the CGN was
provided, however, an explanation of how the molecular
weights and purity were ascertained was not included and the
BMP, TGFβ (Hh?)
proliferation
differentiation
Wnt
Panethcells Stem
cells
ISEMF
(A) (B)
Figure 10. Wnt and BMP signaling roles in the gastrointestinal tract. In the small intestine, cell proliferation and maturation in the crypt–villus unit istightly controlled by Wnt signaling and bone morphogenic protein (BMP) signaling pathways. Wnt controls cell proliferation and early differentiation,while BMP signaling controls differentiation and maturation. The combined process is called epithelial-renewal (B) Gregorieff et al. (2012).
??
β
?
?
??
Normal signaling(A)
- BMP signaling maintains homeostasis
β-catenin
NOTCH
Mesenchyme Epithelium
Loss of mesenchymal TGFβ/BMP signaling(B)
β-catenin
NOTCH - Aberrant epithelial proliferation and polyposis
Mesenchyme Epithelium
(C) Loss of epithelial BMP signaling
Mesenchyme Epithelium
- Increased epithelial proliferation- Lineage maturation defect
β-catenin
NOTCH
Figure 11. Interruption of BMP and Wnt signaling. Normal signalingprocesses are intact (A) and homeostasis in the crypt-villus unit ismaintained. Inhibition of chemical events in mesenchymal tissue causesunscheduled epithelial cell proliferation (B). If epithelial BMP signalingis inhibited the result is an increase in cell proliferation and defects inmaturation (C). Source: Shoyer et al. (2007) Gastroenterology133(3):1036.
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source and molecular weight range of the DS used by
Bhattacharyya et al. (2007) was not reported. Figure 12(C)
shows the effects of �-CGN (LCG) on secreted BMP4
following an exposure time of 1–8 d. These data indicate that
exposure to CGN in drinking water resulted in a reduction in
the secretion of BMP4. There was no indication of a time or
concentration response in this experiment. Without this
information, it is difficult to conclude that the cause of the
reduction was due to CGN (Bhattacharyya et al., 2007). One
possible explanation is that in this test system the larger CGN
molecule interacts with the NCM460 cells by association with
the cell membrane. It is also possible that the CGN binds
tightly to the serum protein present in the culture medium and
is unavailable for interacting with the cells. In this case, the
biochemical effects observed may be due to contaminants in
the culture system or the CGN preparation itself. Regardless,
it is necessary to establish a time or concentration response
for these data before they can be used to explain CGN effects.
In order to determine whether the changes in BMP4
observed in cells are biologically relevant to or representative
of the regulation of BMP4 in the intact rodent or human GI
tract, it is important to review how this signaling pathway
works in vivo. BMP4 plays an important role in many cellular
processes including human colonic epithelial cell renewal,
proliferation, and differentiation (Kosinski et al., 2007) and is
highly controlled at the level of transcription and post-
translational regulation (Sun et al., 2006). Secretion of the
active mature form of BMP4 is essential for its action on cells
in a microenvironment. However, cellular control over the
synthesis, activation, and secretion is highly regulated
(Sun et al., 2006). BMP4 precursor molecule can be
sequestered by a protein in the DAN family, known as
Gremlin, and this binding prevents secretion. This control
mechanism also has been shown to exist in the basal crypt
of colon intestinal EC (Kosinski et al., 2007) (Figure 13).
The complex nature of intestinal signaling pathways that
control cell differentiation from stem cells to terminally
differentiated intestinal EC varies within the crypt. Therefore,
the effects of dietary substances, such as CGN on this
complex pathway, must take into account the entire system
and not a single component.
Clearly, the levels of BMP4 inside the NCM460 cells
relative to the secreted BMP4 are important for under-
standing the true impact of test agents. The signaling and
cellular complexity that exists in the colon crypt in vivo
does not exist in the NCM460 cells in vitro because the
progression from stem cell to differentiated cell is not
present. Therefore, the suppression of active BMP4
secretion reported by Bhattacharyya et al. (2007) provides
data for a hypothesis, but does not provide a clear link
between the in vitro observations and a mechanism for
predicting hazard in humans. To test the hypothesis that
CGN interferes with BMP4 signaling, an animal model
should be used and the analysis should include immuno-
histochemical, siRNA knock out, or other means of
quantifying this endpoint. Without a common mechanism
that links events observed in vitro to actual effects in vivo,
it is not possible to extrapolate the cell-based findings to
adverse events in the human colon. However, the data can
be used to develop mechanism based hypotheses, which
can be tested both in animals and in other in vitro models.
Like all cell models, the limitations of the model must be
addressed in order for the data obtained to be valuable
with respect to identifying human hazard and risk.
Days Post Treatment
BM
P4(
Den
sito
met
ric U
nit)
15000
12000
8000
4000
0
Cn Cg
d2
d1 d2 d4 d6 d8
d4 d6
10
8
6
4
2
0Cn LCG KCG K4 K14 DS CS
BM
P4(
ng/m
g P
rote
in)
Day2
Day4
Percentage (%) Decrease in BMP-4 Secretion with Respect to Control
LCG KCG K4 K14 DS CSDay 2 39.4 14.1 8.5 12.4 47.8 1.8Day 4 41.7 20.0 14.1 17.4 49.1 3.0
ControlLCG10
8
6
4
2
0
BM
P4(
ng/m
g P
rote
in)
(A)
(B)
(C)
Days Post Treatment
Figure 12. Effects of �-CGN, k-CGN, degraded k-CGN, and DS onBMP4 secretion from NCM460 cells. In this experiment, the normalhuman colonic cell line (NCM460) was used as the test system. Theculture medium contained 10% FBS. The experiment exposed cells to1mg/mL �-CGN for 2, 4, or 6 d (A). BMP4 in spent medium wasdetermined using a cytokine antibody array. These data indicatesuppression of BMP4 relative to controls when the data were expressedas raw densitometric units. In panels B and C, the cells were exposed tosingle concentrations of 1mg/mL �-CGN (LCG), 3 mg/mL k-CGN(KCG), 3 mg/mL degraded k-CGN (K4 or K14), 0.8 mg/mL dextransulfate sodium salt (DS), or chondroitin sulfate (CS). An exposureconcentration could not be found. Exposures were for 2 d (white bars, B)or 4 d (black bars, B). When the BMP4 in medium was normalized toprotein, the highest suppression in BMP4 was observed following LCGand DS exposure, but this was not time dependent (B). The lack of timedependency was shown more clearly in panel C. A key point notaddressed in these experiments is the identity and purity of the CGN andthe use of 10% serum in the medium. High and low molecular weightCGN have a high binding affinity for protein and at the lowconcentrations used in this experiment essentially all the CGN wouldbe tightly bound and unavailable for interaction with cells. Source:Bhattacharyya et al. (2007) Dig Dis Sci 52(10):2770.
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The study also measured the Wnt pathway-controlled
mediator protein b-catenin in cell lysate (Bhattacharyya et al.,
2007). There was a modest (37%) increase in this protein,
but a time-dependent change was not observed under the
conditions studied. The study concludes that exposure to
CGN increases b-catenin and reduces BMP4 protein in vitro.
The implication in vivo would be that the balance maintained
between Wnt and BMP4 signaling pathways could be
disrupted resulting in excessive cell proliferation leading to
intestinal polyps. These in vitro findings do not reflect what
has been observed in vivo, where it was shown that no GI
lesions were found in animals exposed to high levels of CGN
in the diet for 90 d (Weiner et al., 2007, and others, see also
Weiner, PART II, 2014). Dextran sulfate sodium salt caused
the same suppression of BMP4 secretion; however, its effect
on b-catenin was not reported, but which has been shown by
others to cause ulcerations of the GI tract and disruption of
BMP4 signaling (Podolsky, 2000).
It is important to note that although the Wnt/BMP
signaling pathways have been implicated in intestinal polyp
formation; this is most often associated with a mutation
occurring in key proteins in these pathways and not to
chemical perturbation (Kolligs et al., 2002; Morrin et al.,
1997). CGN has been shown to be non-genotoxic and not
mutagenic (Mori et al., 1984; see Weiner, Part II, 2014) and
would not be expected to induce mutations in vivo.
In addition, given the large database of animal safety studies
where CGN was administered in diet without intestinal
effects, it is difficult to establish a causative relationship
between in vitro mechanistic data and in vivo effects.
All the signaling pathways investigated (TLR4, insulin
receptor, Wnt, and BMP) are initiated by a ligand outside of
the cell that binds to surface receptors. The in vitro work
discussed here does not establish a concentration–response or
direct and specific binding to these receptors. It is conceivable
that high molecular weight molecules do not interact specif-
ically with these receptors, but may simply coat them under
in vitro conditions or that other impurities carried along with
CGN processing, are producing the in vitro effects reported.
Remember that food-grade CGN is manufactured in a manner
GREM1GREM2CHRDL1
WNT
BMP
Stem cells
MuscularismucosaeBMP antagonists
BMP pathwayBMP1, 2, 5, 7BMPR2, SMAD7NOTCH pathwayJAG1WNT pathwayWNT5B, APC, TCF4Eph/ephrin pathwayEFNA1, EFNB2EPHA2, A5Myc networkMAD, MAX, MXI1
BMP pathwayGREM1, 2CHRDL1NOTCH pathwayNOTCH 1, 2, 3RBPSUH, TLE2WNT pathwayFZD2, 3, 7, B, TCF3,DKK3, SFRP1, 2Eph/ephrin pathwayEPHA1, 4, 7EPHB1, 2, 3, 4, 6Myc networkMYC
DifferentiativeCompartment(active BMP signaling)
ProliferativeCompartment(active WNT signaling)
Stem Cell Niche(ISEMF + SMC providesource of BMP antagonists)
Figure 13. Importance of Wnt and BMP signaling pathways in colon cells. Wnt- and BMP-mediated signaling is essential for maintaining cellhomeostasis in the crypt-villus unit of the small intestine. It is equally important to realize that these important signaling pathways play a similarhomeostatic role in colon epithelial cell development. Once cells have matured, it becomes more difficult to evaluate chemical inhibition or stimulationof these signaling pathways. Clearly, there are many unique genes involved with different roles depending on what stage the cell has entered. Becausethe fully differentiated mature normal human colonic cell line (NCM460), used as a test system in many laboratories, may not possess all these keysignaling processes, each pathway and its function should be characterized prior to using these cells to define chemical effects. Source: Kosinski et al.(2007) Proc Natl Acad Sci 104:15422.
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that ensures the average molecular weight (e.g. 200 000–
800 000 Da), while reducing heavy metals and microbes to
acceptable and safe levels. Furthermore, it is important
to understand the unique properties of CGN that enables
it to bind tightly to proteins.
The number of sulfate groups on CGN is directly
proportional to its size; hence, low molecular weight com-
ponents have fewer sulfate units than high molecular weight
CGN, but the charge density is the same for CGN and
poligeenan. Thus, both will bind to protein. It is the sulfate
moieties that provide the tight binding properties of CGN to
proteins in food. It is this very property that makes CGN an
excellent stabilizer in food products (Blakemore & Harpell,
2010). While it is true that poligeenan would also bind to
proteins, it is important to remember that poligeenan is not
intentionally added to food and in the animal studies where
poligeenan was administered in drinking water the opportun-
ity to bind protein was greatly reduced or absent. This non-
specific or indirect effect would not be expected to occur
in vivo because CGN that is formulated in diet is tightly
associated with protein and as a result is not readily absorbed
from the GI tract. Finally, food increases gastric motility,
which in turn creates a faster transit time. All these factors
would reduce dietary CGN contact with intestinal cells
(Blakemore & Harpell, 2010).
Effects of CGN identified with various cell lines
The biological effects of CGN or poligeenan have been
evaluated in human intestinal EC (NCM460), human hepa-
toma cells (HepG2), human peripheral blood monocytes
(THP-1), Crandall Rees feline kidney cells (CRFK), and
mouse fibroblasts cell models (Benard et al., 2010;
Bhattacharyya et al., 2010b, 2012; Stiles et al., 2008). Large
polymeric molecules can coat the plasma membrane of cells
grown in culture. Receptors located in the plasma membrane
may appear to be affected; however, this is not a true
biological response unless the full undegraded polymer
actually enters the body/cell. Without this basic pharmacoki-
netic data, the effects measured in vitro may have no
relevance to events in vivo unless one demonstrates that
an indirect effect of CGN in the GI tract is responsible for the
systemic event. Even with this last caveat, it is important
to remember that in vitro studies where parent material is
placed directly on cells and an effect measured is considered
in most instances to be a direct effect. Because of this, the
direct effect observed in cells must be treated with caution
when attempting to relate these events to in vivo effects and
mechanisms.
Cell-cycle arrest, cell proliferation, and cytotoxicity
Early studies with poligeenan in animals reported that there
were effects on the intestinal EC (Marcus & Watt, 1980).
Based on these early studies, the effects of �-, k-, and i-CGN
on cell health were assessed in a human (NCM460) intestinal
cell line (Bhattacharyya et al., 2008a). CGN was obtained
commercially and poligeenan was obtained from a collabor-
ator’s laboratory (Mw55000 Da). The exposure concentra-
tion used to determine the effects of CGN on cell viability
ranged from 1 to 10 mg/L with an exposure time of 48 h.
Incorporation of 5-bromo-2’-deoxyuridine into cells during
S-phase was employed as a means to determine the effects of
CGN on cell proliferation.
For this experiment, the NCM460 cells were exposed
to 1 mg/L �-, k-, and i-CGN (Mw not included) and to
poligeenan (Mw 5000 Da) for 8 d. Cell viability was
determined using ethidium homodimer-1, according to Liu
& Hong (2005). No other cytotoxicity marker was evaluated
(e.g. ATP or a leakage enzyme). The inclusion of other
viability markers is important because without a leakage
marker, the changes in viability could be attributed to reduced
cell proliferation or to effects that would not result in cell
death (McKim, 2010). There was no indication that the
viability data were normalized to loss of cell number or to
protein, which could occur as a result of either cell death or
reduced proliferation. Cell viability was compared to the
concentration of CGN, while cell proliferation was compared
to time. After a 24 h exposure to 1 mg/L or 10 mg/L �-CGN,
cell viability of control cells was 93%, while the viability of
cells exposed to 1 mg/L CGN was 88% and in cells treated
with 10 mg/L CGN, viability was 82%. The effect of �-CGN
on cell proliferation was measured on days 1, 2, 4, 6, and 8
following exposure to 1 mg/L CGN. At day 1, there was no
change in cell proliferation and, after 2 d, there was only an
11% reduction in cell proliferation. These changes in cell
proliferation correspond closely to the reported changes in
cell viability and, hence, may reflect loss of cells not
inhibition of cell proliferation. Cell viability data for 1 mg/L
CGN at 48 h was not shown. Even at the highest exposure
concentration tested (10 mg/L), cell viability was nearly 90%
following a 24 h exposure. However, changes in cell prolif-
eration were not shown at this exposure concentration.
In another study, using CRFK cells, no loss in cell viability
was reported after 24 and 48 h exposures to 250 and 500 mg/L
of commercially available CGN (Stiles et al., 2008). Again,
there was no indication that the commercially obtained CGN
was characterized prior to exposing the test system. Neither a
time course for cell viability could be found, nor could a study
that clearly defined the relationship between in vitro concen-
tration and cell proliferation. Hence, a No Observed Adverse
Effect Level (NOAEL) value for cell viability could not be
determined. This is an important parameter because once this
value is known it can be used to determine the effect of time
on cell health, for example, exposing the cells to the NOAEL
concentration over a range of time points. Cell proliferation in
the absence of cell viability is difficult to interpret, as it is
unclear whether the change in the biomarker for proliferation
was due to a true effect of cell replication, or simply a
decrease in the number of cells due to cell death.
In the study by Bhattacharyya et al. (2008a), a cDNA
microarray was used to screen for genes that might be up- or
down-regulated by CGN. Most investigators agree that the use
of microarrays to identify chemical effects on gene expression
is only a first step in identifying a true chemical gene target.
In order to determine whether the suspected genes identified
in an array are, in fact, part of a mechanism underlying a
biological effect, a definitive study using qRT-PCR and
primers designed to focus on the newly identified target genes
should be done. These experiments would typically include at
least four exposure concentrations in order to verify a
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concentration–response. Any change in gene expression
should be normalized first to housekeeping genes and
second to treatment controls. Because test chemicals can
change the expression of the house-keeping genes used to
normalize the expression of the array genes, it is not
uncommon to include multiple house-keeping genes in an
array to insure a correct normalization and interpretation
of the gene data (Lee et al., 2007). In addition, it is generally
recognized that changes in mRNA that are less than 2.5-fold
relative to non-treated controls may be of low biological
significance even though the change is statistically significant
(Olson, 2006; Tusher et al., 2001). This is because of the
inherent run variability in large microarrays, lack of concen-
tration- or dose–response, difficulty or the absence of
normalization to housekeeping genes, and biological versus
technical replicates. In cell culture experiments in multi-well
plates, biological replicates are typically done by performing
the experiment on multiple days, not by using multiple wells.
Based on these criteria, of the 17 selected genes reported,
only 4 showed induction greater than 2.5-fold and none were
higher than five-fold (Bhattacharyya et al., 2008a). Therefore,
these data are important from the standpoint of identifying
potential pathways of change, but the array data alone should
not be used for hazard identification or risk assessment
related to CGN used as a food additive.
In a separate, but related study, a human monocytic cell
line (THP-1) was used to evaluate the effects of CGN and
poligeenan on cell proliferation (Benard et al., 2010).
Following a 24 h exposure to poligeenan and CGN, the
DNA/RNA of the cells was stained using propidium iodide
under a protocol optimized for flow cytometry. The results
showed that while the poligeenan caused cells to accumulate
in G0/G1 phase, resulting in a concomitant reduction of cells
in S-phase, CGN had no detectable effect on cell proliferation
(Benard et al., 2010; Bhattacharyya et al., 2008a). Thus, in
two studies, one using human intestinal EC (NCM460)
(Bhattacharyya et al., 2008a) and the other using a human
monocytic cell line (THP-1) (Benard et al., 2010), the results
following CGN exposure indicate that CGN causes an
inhibition of cell proliferation. These findings appear incon-
sistent with animal studies, in which an increase in TK
activity, a marker of cell proliferation, was reported (Calvert
& Reicks, 1988; Calvert & Satchithanandam, 1992).
However, the work by Benard et al. (2010) does indicate
that the biological effects of the chemically produced
polygalactan fractions (C10¼Mw of 10 000 Da, C40¼Mw
of 40 000 Da) are more biologically active than the high
molecular weight CGN in this system (Table 2).
Innate mucosal immunity and proinflammatorysignaling in vitro
In order to protect the body, the GI tract maintains an active
immune system associated with its mucosal boundary. The GI
tract contains macrophages, lymphocytes, and other cells
important for an immune response. The GI tract is considered
to be a lymphoid organ and the lymphoid tissue, it contains is
referred to as the gut-associated lymphoid tissue (GALT). The
number of lymphocytes in the GALT is similar to the amount
associated with the spleen. The gut lymphoid cells are
distributed between two primary regions: (1) Peyer’s
patches: which are lymphoid follicles analogous to lymph
nodes that contain B-lymphocytes. Peyer’s patches are
located in the mucosa of the small intestine and extend
inward to the submucosa. The highest concentrations of
Peyer’s patches are found in the ileum. (2) The lamina
propria of the mucosa contains IgA-secreting B-lympho-
cytes. (Nagler-Anderson, 2001). The mediator proteins of
the mucosa-associated lymphoid tissue (MALT) and the B-
cell leukemia/lymphoma (BCL) 10 protein have recently
been shown to be important in the canonical pathway for the
induction of nuclear factor kappa-light-chain-enhancer of
activated B cells (NFkB) (Sagaert et al., 2010). These
proteins are found in B cells which can accumulate in
response to chronic infection or inflammation. The pathway
has been well characterized in B-cell lymphomas, but has
not been shown to be functional in non-B-cell normal tissues
of the GI tract.
Toll-like receptors (TLRs) are a group of receptors that
have an important role in the innate immune response system
in the gut. They are transmembrane protein receptors that
recognize structurally conserved chemicals derived from
microbes. Typically, these receptors reside under primary
barriers, such as skin or intestinal tract mucosa. Thus, TLR
substrates must be able to traverse these natural barrier layers
in order to come in contact with and activate TLRs, which
then activate immune responses. These [toll-like] receptors
are designed to recognize non-self-molecules, such as LPS,
and lipoproteins derived from bacteria. They can also
recognize self-molecules such as lipoproteins, DNA, and
ATP that are released from cells when they undergo necrosis.
These internal ligands are referred to as danger associate
molecular patterns (DAMPs) (Miller et al., 2011).
Ligand activation of TLRs requires cooperative binding
with other proteins. TLR4 recognizes LPS, but the
co-receptors lymphocyte antigen 96 (MD-2), cluster of
Table 2. Degraded CGN induced THP-1 cell cycle arrest in G1 phase.
Concentration(mg/mL) G1 S G2/M
Control 45.2� 0.65 43.8� 0.20 11.1� 1.050.125 46.8� 0.50 43.8� 0.35 9.4� 0.150.25 50.3� 0.10 41.6� 0.25 8.2� 0.15
C10 0.5 53.2� 0.55 39.5� 0.35 7.5� 0.901 60.1� 2.40 30.9� 3.05 9.0� 0.652 62.6� 0.45 28.9� 0.95 8.6� 0.400.125 48.3� 0.10 41.7� 0.20 10.1� 0.350.25 51.7� 1.10 39.4� 0.70 8.9� 0.40
C40 0.5 57.2� 2.45 35.1� 1.50 7.7� 0.901 60.4� 3.05 31.4� 3.75 8.3� 0.652 64.2� 4.15 26.5� 5.05 9.4� 0.900.125 47.5� 0.50 44.0� 0.40 8.5� 0.10.25 48.8� 0.25 41.8� 0.35 9.3� 0.80
Native 0.5 50.9� 0.80 39.9� 0.95 9.3� 0.151 52.1� 1.90 37.4� 1.30 10.5� 0.602 50.6� 1.00 40.2� 0.40 9.2� 0.60
From Benard et al. (2010) PLoS One (1):e8666 with permission.THP-1 cells in exponential growth phase were treated with native CGN,
a 10 kDa fragment (C10), and a 40 kDa fragment (C40). Cells wereexposed to the various agents at the concentrations shown in the tablefor 24 h and then stained with propidium iodide. Cell growth wasdetermined using the flow cytometry.
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differentiation 14 (CD14), and LPS-binding protein (LBP)
are required in order to activate downstream signaling
(Figure 14).
TLR4 signaling can be divided into two pathways: one that
is myeloid differentiation primary response gene (88)
(myD88)-dependent and on that is MyD88-independent
(Figure 14). MyD88-mediated signaling results in up-regula-
tion of NFkB.
In most tissues outside of the GI tract, TLR4 is the primary
signaling pathway activated by LPS. Activation of the
classical TLR4 pathway results in the up-regulation of
NFkB which, in turn, controls the expression of several pro-
inflammatory proteins, such as IL-8 and TNF-a (Figure 14).
LPS activation of TLR4 with subsequent expression of NFkB
requires several conditions to occur. First, the cell must
express TLR4 at the membrane surface, and the higher the
receptor density, the more intense the response. Second, LPS
must interact with co-receptor protein MD-2. CD14 and LBP
are necessary for LPS to interact with MD-2 (Figure 14). It is
important to note that not all tissue or cell-lines express TLR4
equally and therefore, LPS response can vary greatly. Some
TLR receptors, like TLR3, can induce the expression of
proteins that suppress the expression of TLR4 proinflamma-
tory proteins. Interferon-beta (INF-b) is one of these negative
feedback proteins.
The GI tract has an active innate immune system that
protects it from pathogen-mediated damage. This immune
mechanism has the ability to recognize unique pathogen-
associated molecular patterns (PAMPs). This is accom-
plished through pattern recognition receptors (PRRs) of
which the family of TLRs are members. Activation of TLRs
leads to an innate immune response which protects the GI
tract from damage. The innate immune response system in
the gut is under tight negative control mechanisms because
hyperactivity can lead to cellular damage (Ishihara et al.,
2010). To this end, in the intestine, there is an important
homeostatic balance that exists with regard to innate
immune-mediated inflammation, mediated by NFkB
(Figure 15). Under normal conditions, the levels of NFkB
are maintained at a physiologic level, but under conditions
TBK1IRAK4
IRAK1
TRAF6
ΙΚΚγ
ΙΚΚα ΙΚΚβIRF3
Ικ−Β
p50 p65
p50 p65IRF3IFN-β
MAP kinase cascade
Membrane
TRIFTIRAP
MyD88TRIF
TRAM TIRAP
TLR3 TLR1 or TLR6 TLR2TLR5-TLR9TLR4CD14
MyD88 MyD88
MyD88-independent MyD88-independent
Figure 14. Intestinal signaling pathways for toll-like receptors. Toll-like receptors (TLR) play a key role in the control of inflammation in many tissues.Activation of these receptors in the GI tract may lead to inflammation. This figure shows how multiple TLRs could induce an inflammatory responsethrough MyD88 independent or dependent pathways. Source: Kosinski et al. (2007) Proc Natl Acad Sci 104:15422.
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of bacterial infection or dysfunction of negative regulators,
NFkB can increase to harmful levels (Ishihara et al., 2006).
The lumen of the GI tract is in constant contact with the
commensal gram negative bacterium that produces LPS.
Without negative feedback mechanisms, chronic exposure to
LPS could lead to induction of proinflammatory molecules,
such as NFkB, IL-8, and TNF-a, which, in turn, could lead
to chronic inflammation and intestinal cell damage
(Figure 15).
The degree of expression, location on the apical or basal
side of the cell, function, and distribution of TLRs along the
GI tract is complex. For example, TLR4 is reduced in apical
membranes and increased in basolateral membranes on
intestinal cells. In the gut, soluble forms of TLR4 and
TLR2 can function as decoy receptors by binding agonists and
preventing their interaction with membrane bound TLR4
(Liew et al., 2005) (Figure 16).
Thus, these receptors provide one of several negative
regulatory mechanisms in the intestinal EC (Ishihara et al.,
2006; Iwami et al., 2000; LeBouder et al., 2003, 2006; Liew
et al., 2005) that protect the GI tract from chronic inflam-
mation. Figure 17 shows the distribution of three TLRs along
the GI tract of mice. Note that TLR4 is expressed at very low
levels in most regions of the intestine, with highest levels in
the distal colon. Given the high concentration of microflora in
the lower colon, the higher abundance of TLR4 functioning in
a protective manner makes sense.
In order to protect itself from the harmful effects of
continual exposure to LPS from gut microflora, the intestinal
EC do not express measurable levels of TLR4 or MD-2
(Abreu et al., 2001, 2002, 2003). In fact, even intestinal cell
lines (Caco-2, T84, and HT-29) show little or no expression of
these proteins. It is tempting to point out that these cell lines
are tumor-derived and, therefore, may not be representative of
in vivo intestinal epithelium. However, TLR4 was also found
in very low levels in normal adult human colonic biopsies of
small intestine resections, and fetal intestinal EC using
immunohistochemical staining and RT-PCR (Abreu et al.,
2002; Cario & Podolsky, 2000; Naik et al., 2001). The
findings by Abreu et al. (2001, 2002) are supported by work
reported by Ishihara et al. (2006) in which the negative
regulatory role of soluble TLR4 with regard to NFkB
expression and its low level of expression in the GI tract
were discussed.
These findings are important because they indicate that the
TLR4-MD-2 signaling pathway for induction of NFkB and
subsequent proinflammatory cytokines could not be a primary
mechanism for inflammation induced by the low amounts of
orally ingested CGN and desensitization of the TLR4
receptors in the GI tract.
In a series of in vitro studies using the human colon derived
cell line (NCM460), the ability of CGN to induce the NFkB
pro-inflammatory response was investigated (Bhattacharyya
et al., 2010b, 2011). The authors demonstrated LPS-mediated
activation in this cell line and detected a modest two-fold
induction of IL-8 in the presence of CGN. A direct link between
TLR4-MyD88 and MALT1–BCL10 activation of NFkB was
shown in connection with CGN exposure. The authors
interpretation of these studies were that oral ingestion of
CGN induces a pro-inflammatory response in intestinal EC via
TLR4 and that this represents a viable hazard for humans that
ingest CGN in their diets (Borthakur et al., 2007, 2012,
Bhattacharyya et al., 2008c, 2010b, 2011). Because concen-
tration response data for TLR4-mediated induction of IL-8
were not demonstrated, it is possible that small amounts of LPS
associated with CGN from commercial sources could be the
reason for the small increase in IL-8. LPS associated with CGN
has been reported by Tsuji et al. (2003). Although the work is
interesting from a mechanistic perspective, it is clear from the
discussions above that the mechanisms measured in vitro may
have little relevance to induction of inflammatory responses.
The in vitro model selected (NCM460 cells) simply does not
Commensals
Mucosal Injury Tolerance
• of TLR (2, 4, 5) pathways
• Pathogen free condition• Antibiotics treatment
Barrier function
• Pathogenic bacterial infection• Drug-stimulation (DS, TNBS, etc.)• Dysfunction of negative regulators• Down-regulation of TLR9-pathway
NF-κBNF-κB
(physiological level)NF-κB
Figure 15. Role of toll-like receptors in the control of intestinal homeostasis. The introduction of foreign chemicals into the GI tract as well as theproduction of LPS by enteric bacteria means that the intestinal cell system must maintain balance among tolerance, mucosal injury, and cellularinflammation. Source: Ishihara et al. (2006) Curr Pharm Des 12(32):4220.
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Figure 16. Diagrammatic representation of intestinal protection mechanisms against inflammation mediated by soluble toll-like receptor (TLR). TLRsare present in the GI-tract and as such, it is possible that ligand activation of these could lead to inflammation. However, it is important to realize thatbecause the environment of the GI tract contains a large amount of enteric bacteria, intestinal cells are repeatedly exposed to cell by-products, such asthe endotoxin lipopolysaacharide (LPS). In order to protect the GI tract from a constant state of inflammation, TLRs have a protective mechanism thatreduces there responsiveness to stimuli. Soluble forms of the receptors are present and serve as decoys that bind ligands, such as LPS, and preventactivation of the inflammatory response. Source: Liew et al. (2005) Nature Rev Immunol 5:451.
Proximal Medial Distal Proximal Medial Distal
ColonSmall IntestineGlandular Stomach
TLR4TLR5TLR2
Figure 17. Distribution of toll-like receptors (TLR) in the gastrointestinal tract. The relative abundance of TLRs in the GI-tract is region specific, withthe highest levels found in the colon. This is also the area of the intestine with the highest concentrations of bacteria. Therefore, LPS stimulatedinflammation would be expected to be least sensitive in this region. This is due to decoy receptors (see Figure 14) which mitigate LPS-likeinflammatory signals. Source: Ishihara et al. (2006) Curr Pharm Des 12(32):4219.
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mimic the necessary mechanisms that are present in the
intestinal tract in vivo.
In addition to the poor correlation between mechanisms
measured in cells and their presence and function in vivo,
there was no clear explanation as to how CGN or poligeenan
actually enters cells. In several in vivo studies in which
CGN was prepared in diet at high doses, there was nearly a
quantitative recovery in feces. More importantly, the dispos-
ition of the small amounts that were not recovered was not
determined. Although some absorption cannot be ruled out, it
is far more likely that the material was still in the GI tract.
Rodents eat their feces, and therefore some of the missing
material may simply have reentered the digestive system. It is
well known that the absorption of macromolecules in the
intestine is very low (Weiner, 1988). Given the high
molecular weights of CGN and poligeenan, both would be
considered macromolecules and as such would be expected to
have a low absorptive capacity.
Immune-related effects reported in animal studies were
observed following either intraperatoneal or intravenous
injections of CGN. There have not been any detailed experi-
ments demonstrating a competitive binding mechanism of
CGN to TLR4 with subsequent activation of downstream
effector molecules in a dose- and time-dependent manner. The
use of known ligands (e.g. LPS) for TLR4 as comparator
molecules has limited value in terms of determining inflam-
mation in vivo, and the identity and purity information of the
CGN used were either omitted or reported inadequately.
Bhattacharyya et al. (2008c) provide data showing that CGN
exposure of NCM460 cells induces the proinflammatory
cytokine IL-8 by activation of the TLR4–MyD88–MALT1–
BCL10-activation of NFkB. However, the maximum increase
in IL-8 protein was two-fold, compared to the nearly 20-fold
increase produced by LPS. If the CGN induction of IL-8 is
mediated by TLR4 interaction, then there should be a
concentration–response relationship. Significant variation in
the purity of CGN purchased from chemical suppliers can
result in inaccurate dosing and uninterpretable results. It would
also be helpful to include poligeenan, as a treatment group to
determine if poligeenan in the test material obtained commer-
cially could have been responsible for the IL-8 induction.
Poligeenan is not found in food-grade CGN, but is known to
produce adverse effects in animals when prepared under
laboratory conditions and administered to animals.
In summary, the mechanism, proposed by Bhattacharyya
et al. (2008c, 2011), for CGN-induced damage to the GI tract
in humans, which is based on in vitro data, may not be
relevant given that LPS exposure in the GI tract in vivo would
not be expected to induce NFkB via TLR4 with the same
potency. In fact, TLR4 is considered to be a negative regulator
of NFkB in the animal and human GI tract, where soluble
forms function as a decoy receptor (Iwami et al., 2000;
LeBouder et al., 2003) that exerts a negative feedback on LPS
mediated pro-inflammatory responses. Thus, it appears that
while the NCM460 line may provide an interesting model for
studying the TLR4 signaling pathway in vitro, extrapolating
these findings directly to human hazard/risk is premature and
can lead to erroneous conclusions.
When chemically produced polygalactan fractions of iota-
CGN with molecular weights of 10 000 and 40 000 Da were
administered in the drinking water (5%) of male Wistar rats
for 55 d, the animals developed pathologies consistent with
intestinal inflammation and these effects were more pro-
nounced with the 40 000 Da fragment (Benard et al., 2010).
It is important to note here that food-grade CGN represents a
spectrum of molecular weights with more than 90% in the
200 000–800 000 Da Mw range and less than 5% making up
the low molecular weight tail. For decades, high doses of
drugs or chemicals have been administered to animals over a
short exposure period in order to assess adverse effects.
However, in most instances, the high dosage represents a
theoretical exposure and, therefore, the data obtained provide
a means of extrapolating the high-dose short exposure time
data to human hazard and risk. It is less correct to chemically
prepare compounds that are not expected to be present in
appreciable amounts following exposure to a parent molecule.
If, however, this approach is taken, in order to ask the
question, what if this molecule was produced at high levels?,
then the data obtained must be placed into context and
extrapolation to human hazard or risk should not occur until
the putative form of the test agent is demonstrated to exist at
appropriate levels in vivo.
Even more care should be exercised when non-drug
compounds like CGN are evaluated. Remember, CGN is not
a drug and is not intended for systemic exposure. In order
to evaluate the effects of various forms of CGN on the
proinflammatory response in vitro, the authors used a human
peripheral blood monocytes and a human monocytic cell
line (THP-1). The cells were exposed to CGN, poligeenan
(10 kDa fragment), and a non-poligeenan CGN fragment
(40 kDa fragment) for 24 h. Following the exposure period,
the levels of TNF-a released were measured in media. In
monocytes isolated from human blood, the results showed a
concentration-dependent increase in TNF-a release by both
fragments of CGN with no statistically significant difference
between the two fragments tested (Figure 18A). Intact CGN
produced no detectable increase in TNF-a release
(Figure 18A). In contrast, when a similar experiment was
conducted with the human monocytic cell line THP-1, there
was an increase in TNF-a induced by both fragments;
however, in this model under the conditions tested (0.005–
1.0 mg/mL), there was no concentration–response. Native
CGN was not included in this experiment (Figure 18C). This
work demonstrates that poligeenan used in this study or the
low molecular weight fragments of CGN can induce a
proinflammatiory response in monocytic cells in vitro.
Whether these in vitro effects were due to receptor binding
at the plasma membrane or to uptake into the cells is
unclear, but these studies reaffirm that high molecular
weight CGN had little effect in these test systems (Benard
et al., 2010). Furthermore, it is unclear whether the low
molecular fragments of CGN were actually the cause of the
experimental observations. This is because fetal or calf
serum (10%) was used to support the growth of both the cell
systems and CGN as well as CGN fragments bind to protein
with high affinity. Therefore, there would not have been any
appreciable amount of free test material to interact with the
cell models.
In a detailed study designed to define regional GI tract
absorption of CS (a glycosaminoglycan (GAG), similar in
DOI: 10.3109/10408444.2013.861797 Food additive carrageenan: Part I 23
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structure to CGN), it was shown that very little intestinal
absorption occurred and that the small amount that did occur
was due to endocytosis in the colon. This was an in vitro study
in which the authors used an organotypic model, consisting of
whole organ culture established by the surgical removal of
specific sections of the digestive tract (Barthe et al., 2004).
14C-labeled CS plus unlabeled CS were used to track
absorption and fast performance liquid chromatography
(FPLC) with a radiometric detector to quantify degradation
products of CS. The molecular weight of the CS was
17 000 Da and was a mixture of both the four and six sulfate
forms. The findings showed that only 9% of the CS was
absorbed by the small intestine, and this was undegraded CS;
17% was absorbed in the cecum and 23% in the colon. The
proportion of degraded CS fragments (disaccharide) increased
in the cecum and colon (Figure 19). CS degradation was due
primarily to microflora present in the colon. Given the
relatively lower molecular weight of CS, compared to CGN,
fragments of CGN or even poligeenan, it is likely that CGN is
not absorbed to any appreciable degree in the upper sections
of the GI tract. The smaller compound CS is used therapeut-
ically to treat osteoarthritis and is taken up by intestinal cells
following oral administration in rats and in dogs (Conte et al.,
1995).
This is also true in humans (Volpi, 2002), however, CS is
broken down to smaller polysaccharide units and absorption is
highly dependent on molecular size (Conte et al., 1995; Volpi,
2002). In the work by Volpi (2002), the highest molecular
weight reported was 14 000 Da. Most of the reported uptake
forms were di- and trisaccharides, where the gut is capable of
absorbing. In contrast, CGN has a much larger molecular size
than CS and appears to be much more resistant to breakdown
in the GI tract. Although it is possible, evidence of CGN
breakdown and subsequent uptake from the GI tract in vivo
has not been clearly shown in animal studies. Finally, while
oral administration of CS has been shown to cause intestinal
inflammation, any uptake of CGN administered in diet is not
associated with inflammatory or other adverse responses
in vivo (Weiner, Part II, 2014).
Effects of CGN on sulfatase activity
Sulfatases are enzymes in the esterase class that catalyze the
hydrolysis of sulfate esters. Sulfate esters can be found on a
300
250
200
150
100
50
00.1 0.5 1.0 0.005 0.01 0.05 0.1 1
Con
trol
TN
F r
elea
se (
pg/m
l)
Nat
ive
dCGN (mg/ml)
(B)
(A)
(D)
(C)
10000
8000
6000
4000
2000
0
TN
F r
elea
se (
pg/m
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Time (hours)
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LPS
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30
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20
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Con
trol
TN
F r
elea
se (
pg/m
l)
LPS
dCGN (mg/ml)
300
250
200
150
100
50
0T
NF
rel
ease
(pg
/ml)
Time (hours)
Control
C10
C40
LPS
Figure 18. Effects of degraded (poligeenan) and low molecular weight pieces of carrageenan on tumor necrosis factor (TNF) release from humanmonocytic cells. Isolated human peripheral monocytic blood cells (A and B) and a human monocytic cell line (THP-1) (C and D) were exposed to avehicle control, native CGN, or to fragments of CGN 10 kDa (gray bars) or 40 kDa (black bars). In isolated human peripheral monocytes, there was aconcentration-dependent increase in TNF release (A) following exposure to both the 10 and 40 kDa fragments. However, native CGN had little effect onTNF release (A), and there is no evidence that native CGN can enter systemic circulation following oral exposure. This observed increase in isolatedhuman peripheral monocytes was also time dependent in terms of TNF release (B). In panel C, THP-1 cells were exposed to various concentrations ofdegraded CGN. However, these cells had little or no concentration response. By comparing the Y-axis of the bar graphs in panels A and C, it is clear thatthe response (panel C) was much less pronounced and was not concentration dependent like the response in Panel (A). These data could indicate thatthe cells are at maximum response under the conditions tested in panel (C) and that at shorter exposure times, a more concentration-dependent responsewould have been observed. However, this does not appear to be the case because LPS over time (panel D) was able to increase TNF release to levels farabove those reported in panel (C). Thus, the THP-1 cells are capable of responding, when a known TLR4 agonist is present, but dCGN does not appearto act by this mechanism in these cells. A key concern with the in vitro data with regard to dCGN is protein binding to the fetal calf serum, which wasadded to the THP-1 cultures. Source: Benard et al. (2010) PLoS One (1):e8666.
24 J. M. McKim Crit Rev Toxicol, Early Online: 1–33
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range of substrates that include steroids, proteins, and
carbohydrates. In animals and humans, sulfatases are found
in the intracellular and extracellular spaces (Prabhu et al.,
2011; Bhattacharyya et al., 2010c). Many of these enzymes
are localized in lysosomes. Lysosomal sulfatases cleave
GAGs and glycolipids. GAGs are long-branched polysacchar-
ides consisting of a repeating disaccharide unit. This carbo-
hydrate is essential to normal development and health. GAGs
can be linked to proteins to form proteoglycans, which, in
turn, form an important part of connective tissues.
Chondroitin 4-sulfate (CS) is an example of a GAG and
can be found in cartilage, tendons, and connective tissues.
Other examples of GAGs are dermatan sulfate and keratan
sulfate.
Several different sulfatases have been identified and
examples include aryl sulfatases A-G (ARSA-G), galactosa-
mine-6-sulfatase (GALN), iduronate-2-sulfatase (IdoA2S),
and steroid sulfatase (STS or ARSC). Aryl-, GALN, and
IdoA2S sulfatases are found in the acidic environment of
lysosomes. STS (ARSC) is found in the endoplasmic reticu-
lum of cells. Substrates highly specific for a particular
sulfatase have been identified and are used to measure activity
of various sulfatases in cell homogenates (Hanson et al.,
2004). ARSB removes the 4-sulfate groups from CS and has
recently been shown to be present in human colonic cell
membranes (Bhattacharyya et al., 2010c), and ARSB appears
to be present in the normal human colon epithelial cell line
(NCM460) (Bhattacharyya & Tobacman, 2007). Although,
this sulfatase has been identified in extracellular spaces, such
as the cell membrane, the overall sulfatase activity between
species and organs has been shown to be quite different
(Rutenburg & Seligman, 1956). In this early work, arylsulfa-
tase (ARS) activity was evaluated in 17 different tissues from
hamster, rat, human, mouse, dog, guinea pig, and rabbit. In
the rat, ARS activity in the colon and small intestine was very
low, while in both human and mouse, activity was undetect-
able in these organs. Immunohistochemical staining for
ARSB in normal human colonic tissue shows extensive
ARSB in the cell membrane, nuclei, and cytoplasm (Prabhu
et al., 2011). A recent comparison of ARSB activity and
protein levels across species and organs has not been done to
our knowledge.
In a recent study by Bhattacharyya & Tobacman (2012),
using the normal human intestinal epithelial cell line
(NCM460), it was reported that k-, �-, and i-CGN can
induce inflammation via production of reactive oxygen
species (ROS), defined as superoxide anion or by activation
of TLR4 receptor signaling and subsequent activation of
NF-kB. Data reported in this study showed that k-CGN can
increase the formation of ROS in NCM460 cells in a
concentration-dependent manner (Figure 20A and B). ROS
was defined as the production of superoxide anion and was
measured with the fluorescent dye, hydroethidine. Two
central experiments were performed: the first was to divide
the three commercially used forms of CGN �, k, and i-CGN
into two treatment groups. One group of CGN was added to
normal human colonic (NCM460) cells at a concentration of
1 mg/L for 24 h, while the other group consisted of three CGN
forms (1 mg/L) each of which was preincubated with human
recombinant arylsulfatase B (rhARSB 1 mg/L at 37 �C) for
18 h and then added to the cell model and incubated for 24 h.
Changes in the amount of ROS formed were compared to
untreated control cells. The results showed that all the
undigested CGN forms increase ROS relative to controls. �-
and i-CGN preincubated with rhARSB also increased ROS
levels; however, k-CGN exposed to sulfatase decreased ROS
levels (Figure 20A). The increase in ROS production was
concentration-dependent following exposure of the cells to
either fully sulfated CGN (black bars) or to CGN preincu-
bated with rhARSB (gray bars) over concentrations ranging
from 0.1 to 5 mg/L (Figure 20B). When CGN was
preincubated with rhARSB and then added to the cells,
there was a reduction in ROS production (Figure 20A and B)
(Bhattacharyya & Tobacman, 2012).
9876543210
% o
f sta
rtin
g ra
dioa
ctiv
ity
Small Intestine
Incubation time (min)0 15 30 45 60 75 90 120
Disaccharide
Initial ACS
181614121086420
% o
f sta
rtin
g ra
dioa
ctiv
ity
Caecum
Incubation time (min)0 15 30 45 60 75 90 120
Disaccharide
Initial ACS
25
20
15
10
5
0
% o
f sta
rtin
g ra
dioa
ctiv
ity
Colon
Incubation time (min)0 15 30 45 60 75 90 120
Disaccharide
Initial ACS
Figure 19. Absorption of chondroitin sulfate over time in rat smallintestine, cecum, and colon. Gastrointestinal tissues were excised fromrats and used for this experiment. Absorption across various regions ofisolated intestine was assessed and is presented above. Chondroitinsulfate (ACS) at 6 mg/mL was used in the intestinal preparations.Exposure times were for 2 h, and the graphs depict the distributionbetween the disaccharides and the original product as detected byFPLC. The data indicate that most degradation occurred in the colon.Source: Barthe et al. (2004) Arzneimittelforschung 54(5):290.
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k-CGN is similar in structure to CS and, as such, it is
recognized as a substrate by ARSB (Figure 20A and B).
Administration of CS to NCM460 cells did not produce the
increase in ROS or in IL-8 secretion observed following CGN
treatment. When k-CGN and CS were co-incubated with
ARSB, and the reaction mixture used to expose cells, it
appeared that CS and k-CGN competed for ARSB binding
sites, as indicated by changes in ROS levels. The removal of
the sulfate moieties reduced CGN’s ability to induced ROS
levels (Bhattacharyya et al., 2012). The implication is that
k-CGN is a substrate and competitive inhibitor of ARSB and
that metabolism via removal of sulfate alters the biological
effects of k-CGN in NCM460 cells. These findings indicate
that the presence and location of the sulfate groups on CGN
impart a molecular signature that is important for recognition
by sulfatase enzymes.
One stated conclusion from this study is that CGN can
competitively inhibit ARSB; however, this conclusion was
based on indirect data. Experiments demonstrating direct
inhibition of ARSB with CGN, CS, and DS at multiple
concentrations were not performed. This information is
necessary in order to ascertain compound interaction with
the enzyme and to measure direct effects on ARSB activity
for each putative substrate (CS and CGN) at different
concentrations. This is a standard approach for identifying
enzyme activity toward a substrate, determining Michalis
Menton kinetic parameters, and for understanding the type of
inhibition (Nussbaumer & Billich, 2005). Figure 21 is an
example of a sulfatase inhibition experiment done at different
concentrations of substrate over time. This type of experiment
provides important kinetic data that could then be used to
determine inhibition, relative to other polysaccharides, and
to assess in vivo effects. The experiments presented here do
demonstrate that k-CGN could be a substrate, provided it
reaches ASRB in tissues in vivo. Whether or not the low
concentration of CGN in food would actually alter normal
metabolism or ASRB substrates cannot be determined from
these experiments.
The data from this work indicate that �-, k-, and i-CGN all
increase ROS in NCM460 cells. k-CGN is a substrate for
ARSB and removal of sulfate inhibits ROS production. This
becomes a hypothesis to explain k-CGN induced inflamma-
tory responses in the GI tract of animals. However, GI tract
inflammation by food-grade CGN has not been demonstrated
in rodent safety studies (Calvert & Reicks, 1988; Calvert &
Satchithanandam, 1992; Weiner et al., 2007; Wilcox et al.,
1992). The CGN induced inflammation model proposed
(Bhattacharyya & Tobacman, 2012) consists of two activation
pathways, the first pathway includes degradation of k-CGN
by carrageenase and a-1(3,6)- galactosidase followed by
binding of degradation products to TLR4 and activation of
NFkB and induction of IL-8. Although in vitro studies have
demonstrated that CGN is a substrate for these enzymes
1200(A)
(B)
1000
800
600
400
200
0
RO
S (
%C
ontr
ol)
control λCGN KCGN ICGN
w/o ARSB+ ARSB
3000
2500
2000
1500
1000
500
00 0.1 1 5
RO
S P
rodu
ctio
n (%
Con
trol
)
K-CGN (mg/L)
Figure 20. Determining the relationship between sulfate content onCGN and production of reactive oxygen species (ROS) in a normalhuman colonic cell line (NCM460). In this experiment, the role ofsulfation in generation of reactive oxygen species was assessed. TheNCM460 cell line was used as the test system. Cells were exposed toCGNs at 1 mg/L (black bars) or to CGN that had been pretreated witharylsulfatase B (ARSB) prior to exposure (gray bars). Exposure tountreated CGN increased the production of ROS (black bars) relativeto control cells. Pretreatment with ARSB caused a reduction inROS production only for k-CGN (A). The increase in ROS wasconcentration dependent (B). CGN was obtained commercially, andthe identity and purity were not reported. Treatments were done inthe presence of 10% fetal bovine serum. CGN binds with highaffinity to serum proteins and this binding occurs via the sulfategroups. Removal of the sulfate groups would impact protein binding.Source: Bhattacharyya et al. (2011) B. J Nutr Biochem [Epub ahead ofprint].
Time [min]
Res
idua
l Act
ivity
0 5 10 15 20
1
0.1
0.01
0.001
Figure 21. Time- and concentration-dependent inhibition of steroidsulfatase (STS). These data depict a typical evaluation of drug orchemical inhibition of an enzyme. In this case, the inhibitor is an arylsulfamate drug. The graph is included as an excellent example of howto characterize drug or chemical impact on enzyme activity. In this case,inhibition was assessed for STS in placental microsomes. Each curverepresents an exposure concentration (5 mM, black circle, 2.5mM, openupside down triangle, 1.25mM, black diamond, 0.31mM open circles,and 0.16mM, of an aryl sulfamate drug). Data were collected at severaltime points resulting in a time- and concentration-dependent pictureof compound inhibition of STS. It is postulated that CGN interfereswith arylsulfatase activity in vivo. Before CGN can be characterizedas an inhibitor of ARSB, a curve set such as the one shown here.Source: Nussbaumer et al. (2005) Curr Med Chem Anticancer Agents5(5):507–528.
26 J. M. McKim Crit Rev Toxicol, Early Online: 1–33
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(Bhattacharyya et al., 2010a), it is not clear whether these
enzymes are present in rodent or human GI tract cells or in
human microflora. Therefore, although the hypothesis pre-
sented may have relevance in terms of identifying possible
enzymatic degradation pathways, in vitro, there is no clear
link between events observed in the in vitro model to those
predicted to occur in rodents or humans exposed to food-
grade CGN in their diets. A last, but important point is that
in vitro the NCM460 cells are grown in the presence of 10%
serum (InCell, 2007). CGNs bind to serum protein with high
affinity and therefore in cell culture the amount of free CGN
to interact with cell surface receptors would be very low.
Based on the work described above, another study
(Yang et al., 2012) was designed to determine whether
CGNs could inhibit five different sulfatases in several cell
lines. CGN effects on ARSB, ARSA, STS, GALNS, and
IdoA2S sulfatase activities were measured following in vitro
exposure to �-, k- and i-CGNs (1 mg/mL) for 4 d. Four
intestinal epithelial cell types (NCM460, CaCO2, T84, and
primary human colonocytes) and six mammary cell types
(MCF-7, MCF-10A, primary myoepithelial cells (MEC),
T47D, HCC1937, and primary mammary EC) were used in
this study. In the NCM460 cells, �-, k-, and i-CGNs were
tested and all three CGNs inhibited all of the sulfatases tested,
to nearly an identical degree (Yang et al., 2012). The study
concludes that this inhibition could result in a remodeling of
GAG composition and potentially alter tissue structure and
function in animals or humans.
This study is inconclusive because it does not establish a
causative relationship among food-grade CGN, inhibition
of intestinal sulfatases, and subsequent remodeling of GAG,
which could then alter tissue structure and function in humans.
The study by Yang et al. (2012) did not evaluate other sulfate-
containing molecules, such as chondroitin, dermatan, or
keratan as similar macromolecules, that are sulfated and are
therefore likely in vitro substrates of the enzymes evaluated. In
addition, larger polysaccharide molecules, such as pectin, guar
gum, or cellulose, were not included. These additional test
groups would have helped ascertain whether the inhibition of
sulfatases was specific to �-CGN or whether other molecules
with a similar structure might also inhibit this class of enzymes.
The data presented by Yang et al. (2012) focused on the effects
of �-CGN, while the study by Bhattacharyya & Tobacman
(2012) focused on k-CGN. The three forms of CGN have
different molecular conformations and different sulfation
patterns, which impart unique physical and chemical proper-
ties. Therefore, care must be taken when discussing the
biological effects of CGN to be sure that the food-grade CGN
used as a food additive has been correctly identified and
represented in the animal and cell-based studies.
The most obvious means by which a macromolecule can
traverse biological membranes is by endocytosis. Endocytosis
pathways are indeed present and functional in NCM460,
CaCo2, and MCF7 cell cultures (Grant & Donaldson, 2009;
Hughson & Hopkins, 1990; Law et al., 2012). However, the
existence of these pathways was demonstrated for proteins
with molecular weights that ranged from only 1600 to
26 000 Da. Therefore, it is conceivable that poligeenan or
fragments of CGN that are not poligeenan (Mw 5000–
40 000 Da) could possibly enter the cell via endocytosis, but
to our knowledge, there is no information to support
endocytosis of CGN, low molecular weight fragments of
CGN, or poligeenan.
The use of intestinal-derived cells seems appropriate given
that the intestinal epithelium would come into direct contact
with ingested CGN; however, the use of cell lines derived
from mammary tissues or primary mammary cells implies
that CGN is absorbed from the GI tract, enters portal
circulation, is taken up by the liver, crosses the sinusoidal
membranes, and the hepatic parenchyma cell membranes,
exits the liver, and is then carried by blood to mammary tissue
where it would elicit a biological effect. In order to
extrapolate in vitro findings to in vivo effects and human
hazard/risk, it is important that in vitro studies clearly
demonstrate cell uptake, chemical identity and purity, and
the biochemical events observed must be shown to be
concentration and time dependent.
The importance of protein binding cannot be over
emphasized when CGN is the test article and cell-based
models are used as the test system. CGN binds with high
affinity to serum protein and, therefore, it is likely that in the
presence of 10% fetal bovine serum (FBS) that little if any
CGN is in the free form and available for cellular interaction.
Moreover, cell-based studies must also demonstrate that
the cells are healthy by monitoring a marker for cell viability.
Cell viability was not evaluated in the Yang et al. (2012)
study, and this parameter is important because injured or
dying cells cannot provide reliable biochemical data.
If CGN in the gut inhibits STS, which is present in gut
microflora, then the effect of the CGN could be protective in
terms of estrogen exposure. Most, if not all steroid hormones
are taken up by the liver and conjugated with glucuronic acid
or sulfate by hepatic UDP-glucuronosyl transferases and
sulfotransferases. The conjugated forms enter the bile and are
deposited into the intestine. Because the conjugated forms
have a reduced lipid solubility, their reabsorption is greatly
reduced and systemic exposure to the estrogens is reduced.
Alternatively, the presence of glucuronidases and sulfatases in
gut microflora can deconjugate steroids, resulting in an
increased reabsorption, which in turn would increase systemic
exposure (Van Eldere et al., 1987). The in vitro work
reported by Yang et al. (2012) suggests that oral exposure to
CGN can inhibit these bacterial sulfatases. If these findings
hold true, then it is conceivable that CGN may reduce steroid
exposure.
Relationship among CGN, myoepithelial cells, andmammary carcinoma
A clear understanding of breast tissue structure is important
in order to understand how chemicals ingested orally might
elicit biochemical effects. The mammary gland is composed
of a branching matrix of ductal tissues. These ductal
networks are composed of two types of epithelial cells,
which are embedded in the connective tissue. These include
the myoepithelial cells and the inner layer of luminal
epithelial cells (Figure 22). These cells are encircled by a
continuous layer of cells called the basement membrane
(BM). The myoepithelial cells contribute to the growth
and maintenance of the BM, and their ability to contract is
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due to their myogenic differentiation (Gudjonsson et al.,
2005; Pandey et al., 2010). Structurally, the myoepithelial
cells are attached to the luminal cells by desmosomes
and to the BM by hemidesmosomes. The BM is composed
of collagenous, as well as, lamin, heparin sulfate, proteo-
glycans, and GAGs. Some of these structures are similar to
CGN structure.
Myoepithelial cells form a barrier separating proliferating
epithelial cells from the BM and stroma. It is important to
note that the myoepitheial cells and the BM also function as a
barrier to small molecules. This means that chemicals, as well
as required growth factors and nutrients, must pass through
the BM and the myoepithelial cells in order to reach inside the
duct. Tumor cells that developed inside the duct must also
traverse these two cell layers before they can enter the stroma
(Figure 23). This means that destruction of both the BM and
the myoepithelial layers is a prerequisite to tumor cell
invasion of stromal tissues. It is currently believed that the
myoepithelial cells play an important role in preventing tumor
invasion of surrounding tissues (Pandey et al., 2010; Sopel,
2010) (Figures 23 and 24).
Tobacman et al. (2001) hypothesized that consumption of
CGN in food is correlated with an increased incidence of
breast cancer in the United States. The premise for this work
is that poligeenan has been reported to be a carcinogen in
the GI tract (IARC, 1983) and that CGN in the diet can be
broken down by acid hydrolysis or enzymatic degradation to
poligeenan (Tobacman et al., 2001). In her introduction, she
states ‘‘Carrageenan, a naturally occurring gum derived
from red seaweed, is of special interest because its degraded
form, known as poligeenan, has been identified as a
carcinogen in animal models of intestinal carcinogenesis’’.
Examination of this statement reveals that, although it is true
that poligeenan has been associated with intestinal cancer in
animals, it is incorrect that food-grade CGN is broken down
to any significant degree in the GI tract to poligeenan. To
our knowledge, poligeenan has never been reported or
quantified after treatment of animals with CGN. Thus,
without this information, the basic hypothesis stated cannot
be tested. In addition, there is an important distinction
between data that is statistically correlated versus data where
one event (abdominal fat) actually causes another event
Myoepithelial Cell
LuminalEpithelial Cell
BasementMembrane(BM)
MilkProteins
LuminalSpace
Figure 22. Cross section of a normal mammary gland duct. A diagrammatic representation of normal mammary gland showing key cell types and theirlocations relative to one another. Source: Pandey et al. (2010) Front Biosci 15:227.
LuminalSpace
BasementMembrane
Myoepithelial Cell
LuminalEpithelial Cell
Invasion, Metastasis
Tumor cells
Nutrients, GFs Stroma
Figure 23. Structural relationship among mammary gland duct cells, basement membrane (BM), and the stroma. Source: Pandey et al. (2010) FrontBiosci 15:228.
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(diabetes). They may be closely related, but one may not
cause the other (Frayn, 2000). Causation and correlation are
frequently misused by scientists and even more so by non-
scientists (e.g. consumer groups). Many assume that if two
events are correlated, then they are also connected causally.
Identifying the true cause of a biological event, such as an
increased incidence of breast cancer, is difficult, which is
why it is generally accepted that a chemical causes an event,
only if the event can be linked to the chemical by dose– and
time–response. Thus, there is no scientific evidence that
supports a causal relationship between food-grade CGN
consumption by humans and breast cancer.
The onset, magnitude, and recovery of cell proliferation
can vary between agents, but no clear evidence has been
presented that would indicate that CGN or its degradation
products are direct carcinogens in the GI tract. In contrast,
CGN has been shown to be non-carcinogenic in chronic
animal studies (Rustia et al., 1980). In general, chemicals
that increase cell proliferation can also promote tumor
development, but this is typically after an initiating event by
another chemical has occurred. Dietary fibers and undi-
gested carbohydrates can undergo fermentation in the colon
by microflora. A major site for fermentation is the cecum in
the proximal colon (Mallett et al., 1985). Fermentation
results in the formation of short chain fatty acids, which
decrease the pH, and has been associated with increased cell
proliferation and, in some instances, with colon cancer
(Jacobs, 1987). However, because undigested materials can
increase the dilution of intestinal contents by drawing fluids
into the lumen, some studies have shown that this effectively
protects the intestine from the development of cancer
(Zoran et al., 1997).
The main point is that a statistical analysis relating
common dietary substances, such as wheat bran, oat bran,
pectin, and guar gum, which leads to an increase in mammary
tumors, may result in a good correlation. However, this should
not be construed as causative without extensive mechanistic,
dose– and time–response data. It is clear that the incidence of
breast cancer has increased over the years, but this is most
likely due to increased obesity, diet high in fats, and an
amazing increase in the use of synthetic estrogens. Many of
these are consumed in amounts that far exceed the daily intake
of dietary CGN.
Cell-based experiments designed to evaluate the effects of
�-CGN on breast myoepithelial cells were done by establish-
ing primary cultures of human breast tissue discarded
following reduction mammoplasty (Tobacman, 1997;
Tobacman & Walters, 2001). Once established, these cultures
were treated with a single low (1 mg/mL) concentration of
�-CGN purchased from a commercial supplier. Both studies
demonstrated significant alterations in the myoepithelial cell
structure. However, no determination of cell viability was
reported. In one study, there was a reduction in cell number in
the CGN-treated groups, but it was unclear whether this was
due to inhibition of cell division, as previously reported by
this laboratory, or cell death. There were no error bars on this
graph and no description of the number of technical and
experimental replicates that were performed. Although the
data reported appears correct in terms of CGN treatment and
damage to myoepithelial cells in vitro, there were no clear
assessments made to explain how similar the in vitro model
was, in terms of cellular structure, to the in vivo case.
Therefore, although interesting, these findings should not be
considered evidence of hazard, and there is insufficient data
to determine risk to humans.
Summary and conclusions
The food ingredient, CGN, has been evaluated in a variety
of animal studies throughout the years and is currently
considered safe for use as a food additive by regulatory
agencies worldwide. It is well known that if CGN is injected
into the foot pad of rats that an immune-mediated inflam-
matory response ensues. Adverse events in the GI tract are
StromaBM (Intact)
DamagedMEP cell
Luminal spaceTumorcells
Tumor cells instroma
iii.BM (Degraded)
Proteolyticenzymes
ii.
IRCs
i.
iv.
v.
Figure 24. Degradation of the basement membrane allows tumor cells to infiltrate the stroma. Source: Pandey et al. (2010) Front Biosci 15:231.
DOI: 10.3109/10408444.2013.861797 Food additive carrageenan: Part I 29
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seen if a low molecular weight material, known as
poligeenan, is administered to rodents orally. Care should
be exercised when data from CGN studies in which the route
of administration was not oral and when CGN was not
formulated with food protein are used to justify in vitro
studies designed to understand mechanisms associated with
routes of exposure not intended for humans. Based on the
fact that in vivo studies with CGN in diet do not produce
inflammation in the GI tract, the in vitro data reported
should not be used for the purpose of identifying human
hazard or for risk assessment. The reasons for this are as
follows: first, CGN is a large molecule with low absorption
and therefore low systemic exposure, although it is import-
ant to remember that indirect systemic effects are possible.
Second, GI tract inflammation and toxicity are observed
when poligeenan is administered orally to rodents, but these
effects are not observed at when food-grade CGN is
administered to rodents in diet. It has been inferred that
CGN can be degraded to poligeenan via acid hydrolysis in
the stomach or by enzyme digestion in the intestine or by
the microflora of the gut. However, degradation of food-
grade CGN formulated in diet has not been demonstrated
in vivo. Third, food-grade CGN is not a single molecule, but
rather a range of molecules that span a molecular weight
spectrum and possess an average molecular weight. The low
molecular weight fragments, which represent a very small
percentage of the CGN spectrum, have been present in
several in vivo dietary feeding toxicology studies and no
adverse effects have been reported. Fourth, some in vitro
studies have suggested a link between the CGN and the
incidence of breast cancer and diabetes. If CGN cannot be
absorbed, then CGN cannot have direct systemic effects on
glucose uptake by liver or muscle. Correlating increased
instances of diabetes or breast cancer over time with CGN is
correlative, not causative, data.
Because CGN is well known to bind with high affinity to
serum proteins, the use of serum protein in the in vitro
studies reviewed would have bound most of the CGN
leaving little if any for interaction with cells. The identity
and purity of the CGN obtained from commercial chemical
supply houses must be verified. Concentration- and time-
dependent responses should be established and, in many
studies, these important data were not shown. It is important
to remember that mechanistic safety data obtained from
in vitro models cannot be used for hazard identification or
risk assessment in animals or humans unless the cell-based
model has been shown to possess the same functional
mechanisms that exist in vivo. Therefore, although the
in vitro observations reported and discussed in this review
may be correct in the cell model used, it is unclear whether
CGN, a contaminant, or a laboratory artifact are the actual
causal agents of these effects. Finally, there is no clear link
between the increasing numbers of biochemical events
reported in vitro and adverse effects in animals under the
intended use conditions of CGN.
Acknowledgements
The author would like to thank Mr. William Blakemore,
F.R.S.C. (retired), Myra Weiner, Ph.D., D.A.B.T., Fellow
A.T.S., and Mr. Christopher Sewall for their detailed
technical reviews of this manuscript. Thanks also to Ms.
Eunice Cuirle for reviewing this article and to Ms. Muriel
Reva for editorial comments, review, and expert adminis-
trative assistance.
Declaration of interest
The author of this paper is identified on the cover page. James
McKim is the Founder, Chief Scientific Officer of CeeTox,
Inc., an in vitro toxicology CRO providing advice on
toxicological and risk assessment issues to private firms.
The current review was prepared for FMC Corporation under
a cost reimbursable contract. FMC Corporation is a manu-
facturer of CGN and products containing CGN. The review
strategy, the review of the literature, analyses, and conclusions
reported in this paper are the professional work product of the
author. The FMC Corporation was given the opportunity to
review the paper and offer comments on the paper. Those
comments did not alter the professional opinions of the
author. The author has not appeared in any legal proceedings
related to the findings reported in this paper. The conclusions
drawn are not necessarily those of the FMC Corporation.
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DOI: 10.3109/10408444.2013.861797 Food additive carrageenan: Part I 33
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