food additive carrageenan: part i: a critical review of...

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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http://informahealthcare.com/txc

mailto:[email protected]

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

2 J. M. McKim Crit Rev Toxicol, Early Online: 1–33

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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%.

DOI: 10.3109/10408444.2013.861797 Food additive carrageenan: Part I 3

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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.


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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


(C) Loss of epithelial BMP signaling


- 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


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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

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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.


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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).

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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)

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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]

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0 5 10 15 20

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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.


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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.


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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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